Compositions and methods for counteracting effects of reactive oxygen species and free radicals

ABSTRACT

Peptide compounds and methods for upregulating expression of a gene encoding an antioxidative enzyme, such as superoxide dismutase or catalase, to counteract harmful oxidative effects of reactive oxygen species and other free radicals are described. The peptide compounds may be used to treat or prevent diseases and conditions characterized by undesirable elevation of reactive oxygen species and other free radicals, to upregulate AP-1 gene expression, and to treat pain. The peptide compounds may be used as components of pharmaceuticals and dietary supplements.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/987,659 filed Nov. 11,2004, now pending; which is a continuation of Ser. No. 09/715,763 filedNov. 17, 2000, now U.S. Pat. No. 6,890,896; which claims the benefitunder 35 U.S.C. 119 to U.S. provisional patent application Ser. No.60/166,381, filed Nov. 18, 1999, each of which are hereby incorporatedby reference herein in their entirety.

FIELD OF THE INVENTION

The present invention is in the field of antioxidative compounds, inparticular, pharmaceutical and nutraceutical compounds for use intherapeutic and prophylactic treatments of diseases and conditionscharacterized by undesirable levels of reactive oxygen species and freeradicals.

BACKGROUND TO THE INVENTION

Biological organisms generate harmful reactive oxygen species (ROS) andvarious free radicals in the course of normal metabolic activities oftissues such as brain, heart, lung, and muscle tissue (Halliwell, B. andGutteridge, J. M. C., eds. Free Radicals in Biology and Medicine,(Oxford: Clarendon Press, 1989)). The most reactive and, therefore,toxic ROS and free radicals include the superoxide anion (O₂.⁻, singletoxygen, hydrogen peroxide (H₂O₂), lipid peroxides, peroxinitrite, andhydroxyl radicals. Even a relatively small elevation in ROS or freeradical levels in a cell can be damaging. Likewise, a release orincrease of ROS or free radicals in extracellular fluid can jeopardizethe surrounding tissue and result in tissue destruction and necrosis.Indeed, hydrogen peroxide, which is somewhat less reactive than thesuperoxide anion, is a well known, broad spectrum, antiseptic compound.In eukaryotes, a major source of superoxide anion is the electrontransport system during respiration in the mitochondria. The majority ofthe superoxide anion is generated at the two main sites of accumulationof reducing equivalents, i.e., the ubiquinone-mediated and the NADHdehydrogenase-mediated steps in the electron transport mechanism.Hydrogen peroxide is generated metabolically in the endoplasmicreticulum, in metal-catalyzed oxidations in peroxisomes, in oxidativephosphorylation in mitochondria, and in the cytosolic oxidation ofxanthine (see, for example, Somani et al., “Response of AntioxidantSystem to Physical and Chemical Stress,” In Oxidants, Antioxidants, andFree Radicals, chapter 6, pp. 125-141, Baskin, S. I. and H. Salem, eds.(Taylor & Francis, Washington, D.C., 1997)).

In normal and healthy individuals, several naturally occurringantioxidant defense systems detoxify the various ROS or free radicalsand, thereby, preserve normal cell and tissue integrity and function.These systems of detoxification involve the stepwise conversion of ROSor free radicals to less toxic species by the concerted activities ofcertain antioxidative enzymes. These antioxidative enzymes are membersof a larger class of molecules known as “oxygen radical scavengers” or“lazaroids” that have an ability to scavenge and detoxify ROS and freeradicals. Vitamins A, C, E, and related antioxidant compounds, such asβ-carotene and retinoids, are also members of this larger class. Inhealthy individuals, sufficient levels of antioxidative enzymes andother lazaroids are present both intracellularly and extracellularly toefficiently scavenge sufficient amounts of ROS and free radicals toavoid significant oxidative damage to cells and tissues.

Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase(GSH-Px) are among the most important and studied of the antioxidativeenzymes. These enzymes function in concert to detoxify ROS and freeradicals. SOD is present in virtually all oxygen-respiring organismswhere its major function is the dismutation (breakdown) of superoxideanion to hydrogen peroxide. Hydrogen peroxide, itself, is a highlyreactive and oxidative molecule, which must be further reduced to avoiddamage to cells and tissues. In the presence of the appropriate electronacceptors (hydrogen donors), CAT catalyzes the further reduction ofhydrogen peroxide to water. In the presence of reduced glutathione(GSH), GSH-Px also mediates reduction of hydrogen peroxide to water by aseparate pathway.

Each of the antioxidative enzymes described above can be furthersubdivided into classes. There are three distinct classes of SOD basedon metal ion content: copper-zinc (Cu—Zn), manganese (Mn), and iron(Fe). In mammals, only the Cu—Zn and Mn SOD classes are present.Mammalian tissues contain a cytosolic Cu—Zn SOD, a mitochondrial Mn SOD,and a Cu—Zn SOD referred to as EC-SOD, which is secreted into theextracellular fluid. SOD is able to catalyze the dismutation of thehighly toxic superoxide anion at a rate of 10 million times faster thanthe spontaneous rate (see, Somani et al., p. 126). Although present invirtually all mammalian cells, the highest levels of SOD activity arefound in several major organs of high metabolic activity, i.e., liver,kidney, heart, and lung. Expression of the gene encoding SOD has beencorrelated with tissue oxygenation; high oxygen tension elevates SODbiosynthesis in rats (Toyokuni, S., Pathol. Int., 49: 91-102 (1999)).

CAT is a soluble enzyme present in nearly all mammalian cells, althoughCAT levels can vary widely between tissues and intracellular locations.CAT is present predominately in the peroxisomes (microbodies) in liverand kidney cells and also in the microperoxisomes of other tissues.

There are two distinct classes of GSH-Px: selenium-dependent andselenium independent. Furthermore, GSH-Px species can be found in thecytosol, as a membrane-associated protein, and as a circulating plasmaprotein.

A recognition of the role of ROS and free radicals in a variety ofimportant diseases and drug side effects has grown appreciably overrecent years. Many studies have demonstrated that a large number ofdisease states and harmful side effects of therapeutic drugs are linkedwith a failure of the antioxidant defense system of an individual tokeep up with the rate of generation of ROS and various free radicals(see, for example, Chan et al., Adv. Neurol., 71:271-279 (1996);DiGuiseppi, J. and Fridovich, I., Crit. Rev. Toxicol., 12:315-342(1984)). For example, abnormally high ROS levels have been found underconditions of anoxia elicited by ischemia during a stroke or anoxiagenerated in heart muscle during myocardial infarction (see, forexample, Walton, M. et al., Brain Res. Rev., 29:137-168 (1999);Pulsinelli, W. A. et al., Ann. Neurol., 11: 499-502 (1982); Lucchesi, B.R., Am. J. Cardiol., 65:14I-23I (1990)). In addition, an elevation ofROS and free radicals has also been linked with reperfusion damage afterrenal transplants. Accordingly, an elevation of ROS and free radicalshas been linked with the progression and complications developed in manydiseases, drug treatments, traumas, and degenerative conditionsincluding oxidative stress induced damage with age, Tardive dyskinesia,Parkinson's disease, Huntington's disease, degenerative eye diseases,septic shock, head and spinal cord injuries, Alzheimer's disease,ulcerative colitis, human leukemia and other cancers, and diabetes (see,for example, Ratanis, Pharmaceutical Executive, pp. 74-80 (April 1991)).

One approach to reducing elevated levels of damaging ROS and freeradicals has involved an attempt to increase the levels of antioxidativeenzymes and other lazaroids by administering those agentstherapeutically. As a result, the commercial market for antioxidativeenzymes and other lazaroids is estimated to exceed $1 billion worldwide.Not surprisingly, research and development of various lazaroids astherapeutic agents has become a highly competitive field. Interest indeveloping SOD itself as a therapeutic agent has been especially strong.This is due, in part, to SOD's status as a recognized anti-inflammatoryagent and the belief that SOD might provide a means for penetrating thenonsteroidal, anti-inflammatory drug (NSAID) market as well (Id., at p.74).

Despite many years of focused research effort, the use of SOD and otherlazaroids has not provided a successful prophylactic or therapeutic toolfor addressing the diseases, disorders and other conditions caused by orcharacterized by the generation of ROS and free radicals. Clearly, thereremains a need for additional therapeutics and methods of treatingdiseases and conditions characterized by the destructive effect ofelevated levels of ROS and free radicals.

SUMMARY OF THE INVENTION

The invention described herein solves the problem of how to counteractthe destructive oxidative effect of elevated levels of ROS and freeradicals by providing peptide compounds that stimulate (i.e.,upregulate) expression of genes encoding antioxidative enzymes, such assuperoxide dismutase (SOD) and/or catalase (CAT), to reduce, eliminate,or prevent an undesirable elevation in the levels of ROS and freeradicals in cells and tissues, and to restore age-related reduction ofconstitutive antioxidative enzymes. Furthermore, the peptide compoundsof this invention may have antioxidative activity independent of theirability to stimulate expression of genes encoding antioxidative enzymes.The formulas of the peptide compounds described herein use the standardthree-letter abbreviation for amino acids known in the art.

In one embodiment, the invention provides a peptide compound having theformula:R₁ Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln R₂  (SEQ ID NO:1),wherein R₁ is absent or is an amino terminal capping group and R₂ isabsent or is a carboxy terminal capping group of the peptide compoundand wherein the peptide compound upregulates expression of a geneencoding an antioxidative enzyme.

In another embodiment, the invention provides a peptide compound havingthe formula:R₁ Gln Thr Leu Gln Phe Arg R₂  (SEQ ID NO:2),wherein R₁ is absent or is an amino terminal capping group and R₂ isabsent or is a carboxy terminal capping group of the peptide compoundand wherein the peptide compound upregulates expression of a geneencoding an antioxidative enzyme.

In yet another embodiment, the invention provides a peptide compoundhaving the formula:R₁ Xaa₁ Gly Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ R2  (SEQ ID NO:3),wherein Xaa₁ and Xaa₂ are, independently, aspartic acid or asparagine;R₁ is absent or is an amino terminal capping group of the peptidecompound; Xaa₃ is absent or Gly; Xaa₄ is absent, Asp, or Phe; Xaa₅ isabsent, Ala, or Phe; Xaa₆ is absent or Ala; R₂ is absent or is a carboxyterminal capping group of the peptide compound; and wherein the peptidecompound upregulates expression of a gene encoding an antioxidativeenzyme. A preferred peptide compound according to the formula,upregulates expression of a gene encoding an antioxidative enzyme andcomprises an amino acid sequence selected from the group consisting of:

Asp Gly Asp Asp Gly Asn Asn Gly Asn Asn Gly Asp Asp Gly Asp Gly Asp,(SEQ ID NO: 4) Asp Gly Asp Gly Phe Ala, (SEQ ID NO: 5)Asp Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 6) Asp Gly Asn Gly Asp Phe Ala,(SEQ ID NO: 7) Asn Gly Asn Gly Asp Phe Ala, (SEQ ID NO: 8) andAsn Gly Asp Gly Asp Phe Ala. (SEQ ID NO: 9)

The invention also provides a peptide compound having the formula:R₁ Asn Ser Thr R₂,wherein R₁ is absent or is an amino terminal capping group; R₂ is absentor is a carboxy terminal capping group of the peptide compound; andwherein the peptide compound upregulates expression of a gene encodingan antioxidative enzyme.

In still another embodiment, the invention provides a peptide compoundhaving the formula:R₁ Phe Asp Gln R₂,wherein R₁ is absent or is an amino terminal capping group; R₂ is absentor is a carboxy terminal capping group of the peptide compound; andwherein the peptide compound upregulates expression of a gene encodingan antioxidative enzyme.

In another embodiment, the invention provides a peptide compound havingthe formula:

(SEQ ID NO: 10) R₁ Xaa₁ Xaa₂ Met Thr Leu Thr Gln Pro R₂,wherein Xaa₁ is absent or Ser; Xaa₂ is absent or Lys; R₁ is absent or isan amino terminal capping group; R₂ is absent or is a carboxy terminalcapping group of the peptide compound; and wherein the peptide compoundupregulates expression of a gene encoding an antioxidative enzyme. Apreferred peptide compound according to the formula, upregulatesexpression of a gene encoding an antioxidative enzyme and comprises anamino acid sequence selected from the group consisting of:

Met Thr Leu Thr Gln Pro (SEQ ID NO: 11) andSer Lys Met Thr Leu Thr Gln Pro (SEQ ID NO: 12)

The invention also provides a peptide compound having the formula:R₁ Xaa₁ Xaa₂ Xaa₃ R₂,wherein Xaa₁ is Asp, Asn, Glu, Gln, Thr, or Tyr; Xaa₂ is absent or anyamino acid (i.e., is variable); Xaa₃ is Asp, Asn, Glu, Thr, Ser, Gly, orLeu; R₁ is absent or is an amino terminal capping group and R₂ is absentor is a carboxy terminal capping group of the peptide compound; whereinthe peptide compound upregulates expression of a gene encoding anantioxidative enzyme. Preferably, a peptide compound of the inventioncomprises the above formula wherein Xaa₂ is selected from the groupconsisting of Val, Gly, Glu, and Gln. More preferably, the peptidecompound is selected from the group consisting of:

Asp Gly, Asn Gly, Glu Gly, Gln Gly, Thr Val Ser, Asp Gly Asp, and AsnGly Asn.

In still another embodiment, the invention provides a peptide compoundhaving the formula:R₁ Leu Xaa₁ Xaa₂ R₂,wherein Xaa₁ is any amino acid; Xaa₂ is Gln or Tyr; R₁ is absent or isan amino terminal capping group; R₂ is absent or is a carboxy terminalcapping group of the peptide compound; and wherein the peptide compoundupregulates expression of a gene encoding an antioxidative enzyme.

The invention also provides a peptide compound having the formula:R₁ Met Thr Xaa₁ R₂,wherein Xaa₁ is Asn, Asp, Glu, Thr, or Leu; R₁ is absent or is an aminoterminal capping group; R₂ is absent or is a carboxy terminal cappinggroup of the peptide compound; and wherein the peptide compoundupregulates expression of a gene encoding an antioxidative enzyme.

In a preferred embodiment, a peptide compound of any of the formulasdescribed herein has the R₁ amino terminal capping group. Morepreferably, the R₁ amino terminal capping group is selected from thegroup consisting of a lipoic acid moiety (Lip, in reduced or oxidizedform); a glucose-3-O-glycolic acid moiety (Gga); 1 to 6 lysine residues;1 to 6 arginine residues; an acyl group of the formula R₃—CO—, where COis a carbonyl group, and R₃ is a hydrocarbon chain having from 1 to 25carbon atoms, and preferably 1 to 22 carbon atoms, and where thehydrocarbon chain may be saturated or unsaturated and branched orunbranched; and combinations thereof. More preferably, when the aminoterminal capping group is an acyl group it is acetyl or a fatty acid.Even more preferably, the amino terminal capping group is an acyl groupselected from the group consisting of acetyl, palmitic acid (Palm), anddocosahexaenoic acid (DHA). In another embodiment, the amino terminalcapping group is a peptide consisting of any combination of arginine andlysine wherein the peptide is not less than two amino acids in lengthand not more than six amino acids in length.

Preferred peptide compounds that upregulate expression of a geneencoding an antioxidative enzyme and that are useful in compositions andmethods of the invention include, but are not limited to, those peptidescomprising an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 1) Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln,(SEQ ID NO: 2) Gln Thr Leu Gln Phe Arg, (SEQ ID NO: 13)Glu Thr Leu Gln Phe Arg, (SEQ ID NO: 14)Gln Tyr Ser Ile Gly Gly Pro Gln, (SEQ ID NO: 15)Ser Asp Arg Ser Ala Arg Ser Tyr, (SEQ ID NO: 12)Ser Lys Met Thr Leu Thr Gln Pro, (SEQ ID NO: 13)Met Thr Leu Thr Gln Pro, (SEQ ID NO: 16)Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu, (SEQ ID NO: 6)Asp Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 4) Asp Gly Asp Gly Asp,(SEQ ID NO: 8) Asn Gly Asn Gly Asp Phe Ala, (SEQ ID NO: 17)Asn Gly Asn Gly Asp, (SEQ ID NO: 7) Asp Gly Asn Gly Asp Phe Ala,(SEQ ID NO: 18) Asp Gly Asn Gly Asp, (SEQ ID NO: 9)Asn Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 19) Asn Gly Asp Gly Asp,(SEQ ID NO: 20) Asn Gly Asp Gly, (SEQ ID NO: 5) Asp Gly Asp Gly Phe Ala,(SEQ ID NO: 21) Asn Gly Asn Gly Phe Ala, (SEQ ID NO: 22)Asp Gly Asn Gly Phe Ala, (SEQ ID NO: 23) Asn Gly Asp Gly Phe Ala,Asp Gly Asp, Asn Gly Asn, Asp Gly Asn, Asn GlyAsp, Asn Ser Thr, Phe Asp Gln, Met Thr Leu, MetThr Asp, Met Thr Asn, Met Thr Thr, Met Thr Glu,Met Thr Gln, Thr Val Ser, Leu Thr Gln, Leu ThrGly, Leu Thr Tyr, Asp Gly, Asn Gly, Glu Gly, GlnGly, Glu Ala, Gln Ala, Gln Gly, Asp Ala, and Asn Ala.

Even more preferred peptide compounds that are useful in compositionsand methods of the invention comprise an amino acid sequence selectedfrom the group consisting of Asp Gly Asp, Asp Gly, Thr Val Ser, and GluAla.

In a more preferred embodiment, the invention provides the above-listedpreferred peptide compounds that also have an amino terminal cappinggroup and/or a carboxy terminal capping group. Even more preferred, theamino terminal capping group is selected from a group consisting of areduced or oxidized lipoic acid moiety (Lip), a glucose-3-O-glycolicacid (Gga) moiety, 1 to 6 lysine residues, 1 to 6 arginine residues, anacyl group having the formula R₃—CO—, where CO represents a carbonylgroup and R₃ is a saturated or an unsaturated (mono- or polyunsaturated)hydrocarbon chain having from 1 to 25 carbons, and combinations thereof.Still more preferably, the amino terminal capping group is the R₃—CO—acyl group wherein R₃ is a saturated or unsaturated hydrocarbon chainhaving 1 to 22 carbons. Even more preferably, the amino terminal cappinggroup is the acyl group that is an acetyl group (Ac), palmitic acid(Palm), or docosahexaenoic acid (DHA). In another preferred embodiment,the above-listed preferred peptide compounds have a carboxy terminalcapping group selected from the group consisting of a primary orsecondary amine.

The peptide compounds useful in the compositions and/or methods of theinvention may also be prepared and used as one or more various saltforms, including acetate salts and trifluoroacetic acid salts, dependingon the needs for a particular composition or method.

The invention also provides methods of counteracting the effects of ROSand free radicals in cells and tissues comprising contacting the cellsor tissues with a peptide compound described herein. In a preferredembodiment of the invention, the peptide compounds of the inventionstimulate (upregulate) expression of a gene(s) encoding superoxidedismutase (SOD) and/or catalase (CAT) enzymes, which enzymes are capableof detoxifying ROS and free radicals in cells and tissues of animals,including humans and other mammals. Preferably, gene expression for bothSOD and CAT proteins are upregulated by contacting cells or tissues witha peptide compound of this invention. Treating cells or tissues with apeptide compound described herein may elevate the expression of gene(s)encoding SOD and/or CAT to sufficiently high levels to providesignificantly increased detoxification of ROS and free radicals comparedto untreated cells or tissues.

Patients having a variety of diseases or conditions have been found topossess undesirable levels of ROS and/or free radicals. In a preferredembodiment of the invention, a composition comprising a peptide compounddescribed herein may be used therapeutically to counteract the effectsof ROS and free radicals present in the body and/or prophylactically todecrease or prevent an undesirable elevation in the levels of ROS andfree radicals associated with particular diseases, conditions, drugtreatments, or disorders. Specifically, this invention provides methodsin which a composition comprising a peptide compound described herein isadministered to an individual to treat or prevent a disease or conditionthat is characterized by the generation of toxic levels of ROS or freeradicals, including but not limited to tissue and/or cognitivedegeneration during aging (senescence), senility, Tardive dyskinesia,cerebral ischemia (stroke), myocardial infarct (heart attack), headtrauma, brain and/or spinal cord trauma, reperfusion damage, oxygentoxicity in premature infants, Huntington's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, Alzheimer's disease, diabetes,ulcerative colitis, human leukemia and other cancers characterized byelevation of ROS or free radicals, age-related elevation of ROS or freeradicals, Down syndrome, macular degeneration, cataracts, schizophrenia,epilepsy, septic shock, polytraumatous shock, burn injuries andradiation-induced elevation of ROS and free radicals (includingUV-induced skin damage).

In a particularly preferred embodiment, this invention provides methodsin which a composition comprising a peptide compound described herein isadministered to an individual to lessen or eliminate side effects causedby drug regimens that generate ROS and free radicals. A number of drugshave been found to cause undesirable elevation of levels of ROS or freeradicals as a toxic side effect. Such drugs include doxorubicin,daunorubicn, BCNU (carmustine) and related compounds such as methyl-BCNUand CCNU, and neuroleptics, such as clozapine. As an adjuvant to suchtherapies, the peptide compounds of this invention can be used todecrease the severity of or eliminate these damaging side effects.Accordingly, the peptides of this invention may be administered to treator prevent drug-induced elevation of ROS or free radicals, such asoccurs during treatment with neuroleptic drugs as in Tardive dyskinesia.

In yet another embodiment, the peptide compounds described herein areused as an alternative or adjuvant to nonsteroidal, anti-inflammatorydrugs (NSAIDs) to treat pain from wounds, arthritis, and otherinflammatory conditions in which ROS and free radicals play a role.

The invention also provides a method of therapeutically orprophylactically treating a disease or disorder, other than stroke, in amammal in which there is an abnormally high level of ROS or freeradicals comprising contacting cells of the mammal with a peptidecompound having the formula:R₁ Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu R₂  (SEQ ID NO:16),where R₁ is absent or is an amino terminal capping group and R₂ isabsent or is a carboxy terminal capping group of the peptide compound.Preferably, the method uses the peptide compound where the aminoterminal capping group R₁ is selected from the group consisting of alipoic acid moiety (in an oxidized or reduced form); aglucose-3-O-glycolic acid group; the acyl group, i.e., R₃—CO—, where COrepresents a carbonyl group and R₃ is a saturated or an unsaturated(mono- or polyunsaturated) hydrocarbon chain having from 1 to 25 (andmore preferably 1-22) carbon atoms; 1 to 6 lysine residues; 1 to 6arginine residues; and combinations thereof. More preferably, the methoduses the peptide compound where the amino terminal capping group R₁ isan acetyl group, a glucose-3-O-glycolic acid group, or a fatty acid.Even more preferably, R₁ is acetyl (Ac), palmitic acid (Palm), lipoicacid (Lip), or docosahexacnoic acid (DHA). In another preferredembodiment, the method uses the peptide compound having the carboxyterminal capping group R₂, and, more preferably, wherein R₂ is a primaryor secondary amine.

The invention also provides therapeutic compositions comprising apeptide compound of the invention in a pharmaceutically acceptablebuffer for administration to an individual to eliminate, reduce, orprevent the generation of toxic levels of ROS or free radicals in cellsor tissues.

Another aspect of the invention provides dietary supplement compositions(also referred to as “nutraceuticals”) comprising a natural source,purified composition obtained from an organism (animal, plant, ormicroorganism), which contains or is enriched for an endogenous peptidecompound described herein, which upregulates expression of one or moregenes encoding an antioxidative enzyme, such as SOD and/or CAT in cellsor tissues. Preferably, dietary supplements of the inventionadditionally comprise an exogenously provided peptide compound describedherein. In a more preferred embodiment, a natural source of a purifiedcomposition from an organism used in making dietary supplementcompositions of the invention is green velvet antler from a ruminant,such as deer or elk, or various plant material, such as roots, stems,leaves, flowers, foliage, herbal mixtures, and tea plants.

Certain peptide compounds of the invention may also stimulate orupregulate expression of the gene encoding transcription factor,activator protein 1 (AP-1). AP-1 in turn serves to activatetranscription of various AP-1-dependent genes. Accordingly, theinvention provides a method of activating transcription factor AP-1 andits transmigration to the cell nucleus and/or stimulating orupregulating the expression of the gene encoding AP-1 transcriptionfactor using a peptide compound described herein, other than a peptidecompound having the formula:

R₁ Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu R₂ (SEQ ID NO: 16),wherein R₁ is absent or is any amino terminal capping group as describedherein and R₂ is absent or is any carboxy terminal capping group as thepeptide compound.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show upregulation of superoxide dismutase-1 (SOD-1) mRNAtranscripts of the SOD-1 gene in rat primary cortical cells in culturesincubated for varying amounts of time (0-48 hours) with the peptidecompound CMX-9236 (100 ng/ml) as measured by the RT-PCR method (seetext). FIG. 1A shows the gel electrophoresis of RT-PCR product(transcripts) as a function of incubation time (Hours). Each lane wasloaded with identical amounts of each cDNA produced by the RT-PCR methodfor each time point. This was verified by the use ofglyceraldehyde-3-phosphate dehydrogenase gene transcript (GAPDH, 451base pairs), which is a transcript of a housekeeping internal referencegene. The right-hand lane labeled “Pos” is a positive control ofupregulation in which cortical cell cultures were stimulated with 10μg/ml of the peptide compound for 3 hours, showing a maximum developmentof SOD-1 transcript levels (208 bp). The lane labeled M shows the DNAduplex ladder marker for molecular size. FIG. 1B shows a bar graphdepicting quantitative analysis of the data of the upregulation of SODmRNA. Diagonal line bars indicate SOD-1 data; open bars indicate GAPDHinternal reference data.

FIGS. 2A and 2B show that peptide compound CMX-9236 upregulated SOD-1gene expression in rat primary myocyte cultures. FIG. 2A shows thedose-response data for the effect of CMX-9236 on the pattern of mRNAsynthesis in primary myocyte cultures after a 3-hour incubation with 0,1, 10, or 100 ng/ml of peptide compound. The analysis used the RT-PCRmethod as in FIGS. 1A and 1B. The presence of a band at the region ofthe gel corresponding to 208 base pairs (bp) indicates that SOD-1 wasupregulated. FIG. 2B shows a bar graph depicting quantitative analysisof the data, which indicates that the 10 ng/ml and 100 ng/ml dosesproduced an upregulation of about 6-fold for SOD-1 mRNA transcripts.GAPDH is an internal housekeeping reference transcript (as in FIGS. 1Aand 1B). Diagonal line bars indicate SOD-1 data; open bars indicateGAPDH internal reference data.

FIGS. 3A and 3B show that peptide compound CMX-9967 upregulated thesynthesis of SOD-1 protein in rat brain primary cortical culturesincubated with 0, 10, and 100 ng/ml of the peptide compound for 5 hours.FIG. 3A shows a Western blot containing a band migrating at 34 kDa (themolecular weight of SOD-1), and two lower molecular weight bandscorresponding to smaller components recognized by the anti-SOD-1antibody. FIG. 3B shows a bar graph of the fold-increase in SOD-1protein as a function of dose of CMX-9967 peptide.

FIGS. 4A and 4B show that peptide compound CMX-9236 upregulated catalasemRNA transcripts of the catalase gene in primary rat cortical cellcultures incubated with the peptide compound CMX-9236 (100 ng/ml) forvarying amounts of time (0-48 hours) as measured by the RT-PCR method.FIG. 4A shows the results of using the RT-PCR method as described inFIG. 1 and specific probes for catalase transcripts. GAPDH is aninternal housekeeping standard (451 bp). FIG. 4B shows a bar graph ofthe fold-increase in catalase and GAPDH (internal standard) transcripts(RT-PCR product) as a function of hours of treatment of the cells withCMX-9236. Diagonal line bars indicate catalase data; open bars indicateGAPDH internal reference data.

FIGS. 5A and 5B show that CMX-9963 and CMX-9967 upregulated mRNAtranscripts for both SOD and catalase genes. FIG. 5A shows the resultsof the RT-PCR method to detect SOD and catalase mRNA transcripts in ratprimary cortical cell cultures incubated for 3 hours with 0, 1, 10, and100 ng/ml of CMX-9963 or CMX-9967. Enhanced staining at the positions ofthe 208 and 95 bp regions of the gel corresponding to the correctlengths for the SOD-1 and catalase markers, respectively, were obtained.FIG. 5B shows a bar graph of the quantitative analysis of the dataindicating fold-increase as a function of dose of CMX-9963 or CMX-9967.Horizontal line bars indicate SOD-1 data; diagonal line bars indicatecatalase data; and open bars indicate GAPDH internal reference data.

FIGS. 6A, 6B, and 6C show that CMX-9236 activated transcription factorAP-1 in primary rat cortical cultures stimulated for 3 hours withvarious concentrations (0, 1, 10, 100 ng/ml) of peptide compoundCMX-9236. FIG. 6A shows the dose-response results for AP-1 activationusing the electrophoretic mobility shift assay (EMSA) procedure (seetext). The positions of migration corresponding to c-Jun/c-Fos AP-1heterodimer and to c-Jun/c-Jun AP-1 homodimer are indicated. FIG. 6Bshows a quantitative analysis of the data plotted as fold-increase as afunction of dose of CMX-9236 peptide. FIG. 6C shows results of EMSAs inwhich the specificity of the interaction of the probe for AP-1 isillustrated in probe competition experiments in which non-radiolabeled(cold) AP-1 probe and cold mutant AP-1 probe were added to nuclearextracts prior to P³² probe addition and prior to electrophoresis. Coldprobes were used at 0×, 5×, 25×, 50× molar excess relative to the 0.5pmol of radiolabeled probe. The positions of migration corresponding toc-Jun/c-Fos AP-1 heterodimer and to c-Jun/c-Jun AP-1 homodimer areindicated.

DETAILED DESCRIPTION

This invention is based on the discovery of peptide compounds thatincrease the expression of one or both genes encoding a complementarypair of enzymes, i.e., superoxide dismutase (SOD) and catalase (CAT),which are major components of the antioxidative defense mechanism orsystem in cells and tissues to detoxify reactive oxygen species (ROS)and free radicals. ROS and free radicals are generated during electrontransport and normal respiration and other metabolic processes,including during the metabolism of various drugs, and must be rapidlydetoxified to prevent permanent and continuing damage to cells andtissues. In addition, a number of diseases or conditions, including theaging process (senescence), have also been characterized by an elevationof ROS and/or free radicals to toxic levels that in fact damage cellsand tissues. Accordingly, the peptide compounds described herein arevaluable therapeutic and prophylactic compounds for counteracting thegeneration of harmful levels of ROS and free radicals in an individual.

In order that the invention may be better understood, the followingterms are defined.

Abbreviations: Amino acid residues described herein may be abbreviatedby the conventional three letter or one letter abbreviation know in theart (see, e.g., Lehninger, A. L., Biochemistry, second edition (WorthPublishers, Inc., New York, 1975), p. 72). Other abbreviations usedherein include: “DHA” for a docosahexaenoic acid moiety; “Lip” for alipoic acid moiety; “Palm” for a palmitic acid moiety (i.e., a palmitoylgroup); “Ac” for an acetyl moiety; “Gga” for a glucose-3-O-glycolic acidmoiety; “SOD” for super oxide dismutase; “CAT” for catalase; “GAPDH” forglyceraldehyde-3-phosphate dehydrogenase; and “ROS” for reactive oxygenspecies. Still other abbreviations are indicated as needed elsewhere inthe text.

“Hydrocarbon” refers to either branched or unbranched and saturated orunsaturated hydrocarbon chains. Preferred hydrocarbon chains found insome of the peptide compounds described herein contain between 1 and 25.More preferred are hydrocarbon chains between 1 and 22 carbon atoms.

“Reactive oxygen species” or “ROS”, as understood and used herein,refers to highly reactive and toxic oxygen compounds that are generatedin the course of normal electron transport system during respiration orthat are generated in a disease or during treatment with certaintherapeutic agents for a particular disorder. ROS include, but are notlimited to, the superoxide anion (O₂.⁻), hydrogen peroxide (H₂O₂),singlet oxygen, lipid peroxides, and peroxynitrite.

“Free radical”, as understood and used herein, refers to any atom or anymolecule or compound that possesses an odd (unpaired) electron. By thisdefinition, the superoxide anion is also considered a negatively chargedfree radical. The free radicals of particular interest to this inventionare highly reactive, highly oxidative molecules that are formed orgenerated during normal metabolism, in a diseased state, or duringtreatment with chemotherapeutic drugs. Such free radicals are highlyreactive and capable of causing oxidative damage to molecules, cells andtissues. One of the most common and potentially destructive types of thefree radicals other than the superoxide anion is a hydroxyl radical.Typically, the generation of ROS, such as superoxide anion or singletoxygen, also leads to one or more other harmful free radicals as well.Accordingly, phrases such as “ROS and free radicals” or “ROS and otherfree radicals”, as understood and used herein, are meant to encompassany or all of the entire population of highly reactive, oxidativemolecular species or compounds that may be generated in a particularmetabolic state or condition of cells and tissues of interest (see, forexample, Somani et al, “Response of Antioxidant System To Physical andChemical Stress,” In Oxidants, Antioxidants, and Free Radicals, chapter6: 125-141 (Taylor & Francis, Washington, D.C., 1997)).

“Oxygen radical scavengers” or “lazaroids” are a class of compounds thathave an ability to scavenge and detoxify ROS and free radicals. VitaminsA, C, E, and related antioxidant compounds, such as β-carotene andretinoids, are also members of this large class of compounds, as areantioxidative enzymes, such as SOD and CAT. In healthy individuals,sufficient levels of antioxidative enzymes and other lazaroids arepresent both intracellularly and extracellularly to efficiently scavengesufficient amounts of ROS and free radicals to avoid significantoxidative damage to cells and tissues.

“Peptide compound”, as understood and used herein, refers to anycompound that contains at least one peptide bond. “Peptide compound”includes unmodified or underivatized peptides, typically containingfewer than about 20 amino acids, as well as derivatives of peptides.Derivative or derivatized peptides contain one or more chemical moietiesother than amino acids that are covalently attached at the aminoterminal amino acid residue, the carboxy terminal amino acid residue, orat an internal amino acid residue.

“Natural source purified”, as understood and used herein, describes acomposition of matter purified or extracted from an organism orcollection of organisms occurring in nature or in a cultivated statethat have not been altered genetically by in vitro recombinant nucleicacid technology, including but not limited to animals, any species ofcrops used for beverage and food, species of uncultivated plants growingin nature, species of plants developed from plant breeding, andmicroorganisms that have not been altered genetically by in vitrorecombinant technology. Particularly preferred natural sources forpreparing natural source purified compositions of matter of theinvention are green velvet antler of ruminants, such as deer and cattle,and plant tissue, such as roots, stems, leaves, and flowers from plantsused as herbs and teas.

An “amino terminal capping group” of a peptide compound described hereinis any chemical compound or moiety that is covalently linked orconjugated to the amino terminal amino acid residue of a peptidecompound. The primary purpose of such an amino terminal capping group isto inhibit or prevent intramolecular cyclization or intermolecularpolymerization, to promote transport of the peptide compound across theblood-brain barrier, or to provide a combination of these properties. Apeptide compound of this invention that possesses an amino terminalcapping group may possess other beneficial activities as compared withthe uncapped peptide, such as enhanced efficacy or reduced side effects.For example, several of the amino terminal capping groups used in thepeptide compounds described herein also possess antioxidative activityin their free state (e.g., lipoic acid) and thus, may improve or enhancethe antioxidative activity of the peptide in its uncapped form. Examplesof amino terminal capping groups that are useful in preparing peptidecompounds and compositions according to this invention include, but arenot limited to, 1 to 6 lysine residues, 1 to 6 arginine residues, amixture of arginine and lysine residues ranging from 2 to 6 residues,urethanes, urea compounds, a lipoic acid (“Lip”) or a palmitic acidmoiety (i.e., palmitoyl group, “Palm”), glucose-3-O-glycolic acid moiety(“Gga”), or an acyl group that is covalently linked to the aminoterminal amino acid residue of the peptide. Such acyl groups useful inthe compositions of the invention may have a carbonyl group and ahydrocarbon chain that ranges from one carbon atom (e.g., as in anacetyl moiety) to up to 25 carbons (such as docosahexaenoic acid, “DHA”,which has a hydrocarbon chain that contains 22 carbons). Furthermore,the carbon chain of the acyl group may be saturated, as in a palmiticacid, or unsaturated. It should be understood that when an acid (such asDHA, Palm, or Lip) is present as an amino terminal capping group, theresultant peptide compound is the condensed product of the uncappedpeptide and the acid.

A “carboxy terminal capping group” of a peptide compound describedherein is any chemical compound or moiety that is covalently linked orconjugated to the carboxy terminal amino acid residue of the peptidecompound. The primary purpose of such a carboxy terminal capping groupis to inhibit or prevent intramolecular cyclization or intermolecularpolymerization, to promote transport of the peptide compound across theblood-brain barrier, or to provide a combination of these properties. Apeptide compound of this invention possessing a carboxy terminal cappinggroup may possess other beneficial activities as compared with theuncapped peptide, such as enhanced efficacy, reduced side effects,enhanced hydrophilicity, enhanced hydrophobicity, or enhancedantioxidative activity, e.g., if the carboxy terminal capping moietypossesses a source of reducing potential, such as one or more sulfhydrylgroups. Carboxy terminal capping groups that are particularly useful inthe peptide compounds described herein include primary or secondaryamines that are linked by an amide bond to the t-carboxyl group of thecarboxy terminal amino acid of the peptide compound. Other carboxyterminal capping groups useful in the invention include aliphaticprimary and secondary alcohols and aromatic phenolic derivatives,including flavenoids, with C1 to C26 carbon atoms, which form esterswhen linked to the carboxylic acid group of the carboxy terminal aminoacid residue of a peptide compound described herein.

Peptide compounds of the invention also include any peptide containingmodifications of the side chain of one or more amino acid residueswithin the peptide chain. Such modifications include (withoutlimitation) conservative amino acid substitutions, addition ofprotective or capping groups on reactive moieties, and other changesthat do not adversely destroy the activity of the peptide (i.e., itsantioxidative activity and/or its ability to stimulate expression of agene encoding SOD and/or CAT).

“Radiation”, as understood and used herein, means any type ofpropagating or emitted energy wave or energized particle, includingelectromagnetic radiation, ultraviolet radiation (UV), and othersunlight-induced radiation and radioactive radiation. The effects ofsuch radiation may affect the surface or underlayers of the skin or mayproduce systemic damage at a remote site in the body.

“Upregulate” and “upregulation”, as understood and used herein, refer toan elevation in the level of expression of a gene product in a cell ortissue. The peptide compounds described herein are capable ofupregulating expression of genes encoding superoxide dismutase (SOD),catalase (CAT), and/or AP-1 transcription factor (AP-1) beyond thelevels normally found in cells and tissues that have not been treated(contacted) with the peptide compounds. Thus, an elevation in the levelof SOD, CAT, or AP-1 mRNA transcript; in SOD, CAT, or AP-1 gene product(protein) synthesis; in the level of SOD or CAT enzyme activity, or inthe level of an AP-1 factor dependent transcription activity indicateupregulation of gene expression. Expression of SOD, CAT, and AP-1 genescan be detected by a variety of ways, including Northern blotting todetect mRNA transcripts encoding the enzyme, by Western immunoblottingto detect the gene product, in the case of SOD and CAT, by usingstandard assays for SOD or CAT enzymatic activities, or in the case ofAP-1, by using an AP-1 dependent transcription expression assay.

“Nutraceutical” and “dietary supplement”, as understood and used herein,are synonymous terms, which describe compositions that are prepared andmarketed for sale as non-regulated, orally administered, sources of anutrient and/or other compound that is purported to contain a propertyor activity that may provide a benefit to the health of an individual. Adesirable component compound identified in a dietary supplement isreferred to as a “nutrichemical”. Nutrichemicals may be present in onlytrace amounts and still be a desirable and marketable component of adietary supplement. Commonly known nutrichemicals include trace metals,vitamins, enzymes that have an activity that is considered beneficial tothe health of an individual, and compounds that upregulate such enzymes.Such enzymes include antioxidative enzymes, such as superoxide dismutase(SOD) and catalase (CAT), which counteract the harmful oxidative effectsof reactive oxygen species (ROS) and other free radicals. Accordingly,one or more peptide compounds described herein that is endogenouslypresent and/or added exogenously to a composition manufactured for saleas a dietary supplement is a nutrichemical of that dietary supplement.

Other terms will be evident as used in the following description.

Peptide Compounds and Compositions

The invention provides peptide compounds described herein for use incompositions and/or methods that are not previously described in the artand that are capable of upregulating SOD and/or CAT in eukaryotic cells,which have at least one functional gene encoding the SOD and/or CATenzymes. Upregulating levels of SOD and/or CAT in cells or tissuesprovides an enhanced detoxification system to prevent, reduce, oreliminate the harmful oxidative activity of ROS and free radicals.Preferred peptides and peptide compounds of this invention upregulateboth SOD and CAT. The peptide compounds described herein may alsoupregulate the AP-1 transcription factor, which inter alia may enhanceexpression of antioxidative gene products and/or growth factors.

The peptide compounds provided by the invention are preferably less thanabout 20, and, in order of increasing preference, less than about 18,15, 13, 9, 6, 5, 4 and 3, amino acids in length and are able toupregulate expression of a gene(s) for SOD and/or CAT in cells andtissues. Such activity may be tested in vitro, e.g., in tissue culture.The peptide compounds of the invention show upregulation activity at lowconcentrations, i.e., in the range of nanograms of peptide compound permilliliter (ml). Such high potency is similar to that exhibited byvarious hormones, such as luteinizing hormone releasing hormone (LHRH)or human growth hormone. Accordingly, the peptide compounds describedherein may be prepared, stored, and used employing much of the availabletechnology already applied to the preparation, storage, andadministration of known therapeutic hormone peptides.

The peptide compounds described herein may contain a peptide to whichadditional modifications have been made, such as addition of chemicalmoieties at the amino terminal and/or carboxy terminal amino acidresidues of the peptide, conservative amino acid substitutions ormodifications of side chains of internal amino acid residues of thepeptide that do not destroy the desired activity of the peptide. It hasbeen observed that intramolecular cyclization and some intermolecularpolymerizations of the peptide compounds described herein tend toinactivate or decrease the activity of the peptide compound so that thepeptide compound cannot effectively upregulate SOD, CAT, or AP-1.Accordingly, the most useful peptide compounds are the least susceptibleto cyclization reactions and polymerization or conjugation with otherpeptide compound molecules. In addition to maintaining or enhancing theability of these peptides to upregulate SOD, CAT and/or AP-1, suchmodifications may advantageously confer additional benefits. Forexample, amino terminal capping groups may promote transport of thepeptide compound across the blood-brain barrier (see, for example, PCTpublication WO 99/26620). This property is particularly important when apeptide compound is used to upregulate SOD and CAT in brain tissue andparts of the central nervous system. Amino terminal capping groups thatpromote transport across the blood-brain barrier may also preventcyclization of the peptide compound to which they are attached or mayprevent polymerization with other peptide compounds.

Preferred amino terminal capping groups include a lipoic acid moiety,which can be attached by an amide linkage to the α-amino group of theamino terminal amino acid of a peptide. Lipoic acid (“Lip”) in its freeform possesses independent antioxidative activity and may enhance theantioxidative activity of the peptides of this invention when used as anamino terminal capping group. An amino terminally linked lipoic acidmoiety may be in its reduced form where it contains two sulfhydrylgroups or in its oxidized form in which the sulfhlydryl groups areoxidized and form an intramolecular disulfide bond and, thereby, aheterocyclic ring structure. Another amino terminal capping group usefulin preparing peptide compounds of the invention is aglucose-3-O-glycolic acid moiety (“Gga”), which can be attached in anamide linkage to the α-amino group of the amino terminal amino acid of apeptide compound. The glucose moiety may also contain furthermodifications, such as an alkoxy group replacing one or more of thehydroxyl groups on the glucose moiety.

Another example of an amino terminal capping group useful in the peptidecompounds described herein is an acyl group, which can be attached in anamide linkage to the α-amino group of the amino terminal amino acidresidue of a peptide compound. The acyl group has a carbonyl grouplinked to a saturated or unsaturated (mono- or polyunsaturated),branched or unbranched, hydrocarbon chain of 1 to 25 carbon atoms inlength, and more preferably, the hydrocarbon chain of the acyl group is1 to 22 carbon atoms in length, as in DHA. The acyl group preferably isacetyl or a fatty acid. The fatty acid used as the acyl amino terminalcapping group may contain a hydrocarbon chain that is saturated orunsaturated and that is either branched or unbranched. Preferably thehydrocarbon chain is 1 to 25 carbon atoms in length, and more preferablythe length of the hydrocarbon chain is 1-22 carbon atoms in length. Forexample, fatty acids that are useful as amino terminal capping groupsfor the peptide compounds of this invention include, but are not limitedto: caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0),myristic acid (C14:0), palmitic acid (“Palm”) (C16:0), palmitoleic acid(C16:1), C16:2, stearic acid (C18:0), oleic acid (C18:1), vaccenic acid(C18:1-7), linoleic acid (C18:2-6), α-linolenic acid (C18:3-3),eleostearic acid (C18:3-5), β-linolenic acid (C18:3-6), C18:4-3, gondoicacid (C20:1), C20:2-6, dihomo-γ-linolenic acid (C20:3-6), C20:4-3,arachidonic acid (C20:4-6), eicosapentaenoic acid (C20:5-3), docosenoicacid (C22:1), docosatetraenoic acid (C22:4-6), docosapentaenoic acid(C22:5-6), docosapentaenoic acid (C22:5-3), docosahexaenoic acid (“DHA”)(C22:6-3), and nervonic acid (C24: 1-9). Particularly preferred fattyacids used as acyl amino terminal capping groups for the peptidecompounds described herein are palmitic acid (Palm) and docosahexaenoicacid (DHA). DHA and various other fatty acid moieties appear to promotetransport of molecules to which they are linked across the blood-barrier(see, for example, PCT publication WO 99/40112 and PCT publication WO99/26620). Accordingly, such fatty acyl moieties are particularlypreferred when a peptide compound described herein will be administeredto counteract the oxidative effects of ROS and free radicals in braintissue and/or other parts of the central nervous system.

In addition, in certain cases the amino terminal capping group may be alysine residue or a polylysine peptide, preferably where the polylysinepeptide consists of two, three, four, five or six lysine residues, whichcan prevent cyclization, crosslinking, or polymerization of the peptidecompound. Longer polylysine peptides may also be used. Another aminoterminal capping group that may be used in the peptide compoundsdescribed herein is an arginine residue or a polyarginine peptide,preferably where the polyarginine peptide consists of two, three, four,five, or six arginine residues, although longer polyarginine peptidesmay also be used. An amino terminal capping group of the peptidecompounds described herein may also be a peptide containing both lysineand arginine, preferably where the lysine and arginine containingpeptide is two, three, four, five or six residue combinations of the twoamino acids in any order, although longer peptides that contain lysineand arginine may also be used. Lysine and arginine containing peptidesused as amino terminal capping groups in the peptide compounds describedherein may be conveniently incorporated into whatever process is used tosynthesize the peptide compounds to yield the derivatized peptidecompound containing the amino terminal capping group.

The peptide compounds useful in the compositions and methods of theinvention may contain a carboxy terminal capping group. The primarypurpose of this group is to prevent intramolecular cyclization orinactivating intermolecular crosslinking or polymerization. However, asnoted above, a carboxy terminal capping group may provide additionalbenefits to the peptide compound, such as enhanced efficacy, reducedside effects, enhanced antioxidative activity, and/or other desirablebiochemical properties. An example of such a useful carboxy terminalcapping group is a primary or secondary amine in an amide linkage to thecarboxy terminal amino acid residue. Such amines may be added to theα-carboxyl group of the carboxy terminal amino acid of the peptide usingstandard amidation chemistry.

The peptide compounds used in the compositions and methods of theinvention may contain amino acids with charged side chains, i.e., acidicand basic amino acids. Most preferably, if a peptide compound containscharged amino acids, then the charged amino acids are either all acidicamino acids, i.e., negatively charged, or are all basic amino acids,i.e., positively charged. Such uniformity in charged amino acidscontributes to stability of the peptide compounds and prevents theformation of cyclic, crosslinked or polymerized forms of a peptidecompound during storage or during use in vivo. Cyclization,crosslinking, or polymerization of a peptide compound described hereinmay abolish all or so much of the activity of the peptide compound sothat it cannot be used in the therapeutic or prophylactic compositionsand methods of the invention. Furthermore, some cyclic peptide compoundsare potentially toxic. Accordingly, if a peptide compound contains basic(positively charged) amino acid residues, then it is recommended thatthe carboxy terminal carboxylic acid group be converted to an amide(i.e., by use of a carboxy terminal capping group) to prevent thecarboxylic acid group from reacting with a free amino group in the samepeptide compound to form a cyclic compound or in a different peptidecompound to form a polymerized or crosslinked peptide compound.

In addition, peptide compounds described herein may contain one or moreD-amino acid residues in place of one or more L-amino acid residuesprovided that the incorporation of the one or more D-amino acids doesnot abolish all or so much of the activity of the peptide compound thatit cannot be used in the compositions and methods of the invention.Incorporating D-amino acids in place of L-amino acids may advantageouslyprovide additional stability to a peptide compound, especially in vivo.

The peptide compounds can be made using standard methods or obtainedfrom a commercial source. Direct synthesis of the peptides of thepeptide compounds of the invention may be accomplished usingconventional techniques, including solid-phase peptide synthesis,solution-phase synthesis, etc. Peptides may also be synthesized usingvarious recombinant nucleic acid technologies, however, given theirrelatively small size and the state of direct peptide synthesistechnology, a direct synthesis is preferred and solid-phase synthesis ismost preferred. In solid-phase synthesis, for example, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the α-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents, and reactionconditions used throughout the synthesis and are removable underconditions, which do not affect the final peptide product. Stepwisesynthesis of the polypeptide is carried out by the removal of theN-protecting group from the initial (i.e., carboxy terminal) amino acid,and coupling thereto of the carboxyl end of the next amino acid in thesequence of the polypeptide. This amino acid is also suitably protected.The carboxyl group of the incoming amino acid can be activated to reactwith the N-terminus of the bound amino acid by formation into a reactivegroup such as formation into a carbodimide, a symmetric acid anhydride,or an “active ester” group such as hydroxybenzotriazole orpentafluorophenyl esters. The preferred solid-phase peptide synthesismethods include the BOC method, which utilizes tert-butyloxycarbonyl asthe α-amino protecting group, and the FMOC method, which utilizes9-fluorenylmethloxycarbonyl to protect the α-amino of the amino acidresidues, both methods of which are well-known by those of skill in theart (see, Stewart et al., Solid-Phase Peptide Synthesis (W.H. FreemanCo., San Francisco 1989); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963); Bodanszky and Bodanszky, The Practice of Peptide Synthesis(Springer-Verlag, New York 1984), incorporated herein by reference).

Peptide compounds according to the invention may also be preparedcommercially by companies providing peptide synthesis as a service(e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; AnaSpec, Inc., SanJose, Calif.). Automated peptide synthesis machines, such asmanufactured by Perkin-Elmer Applied Biosystems, also are available.

Peptide compounds useful in the compositions and methods of theinvention may also be prepared and used in a salt form. Typically, asalt form of a peptide compound will exist by adjusting the pH of acomposition comprising the peptide compound with an acid or base in thepresence of one or more ions that serve as counter ion to the net ioniccharge of the peptide compound at the particular pH. Various salt formsof the peptide compounds described herein may also be formed orinterchanged by any of the various methods known in the art, e.g., byusing various ion exchange chromatography methods. Cationic counter ionsthat may be used in the compositions described herein include, but arenot limited to, amines, such as ammonium ion; metal ions, especiallymonovalent, divalent, or trivalent ions of alkali metals (e.g., sodium,potassium, lithium, cesium), alkaline earth metals (e.g., calcium,magnesium, barium), transition metals (e.g., iron, manganese, zinc,cadmium, molybdenum), other metals (e.g., aluminum); and combinationsthereof. Anionic counter ions that may be used in the compositionsdescribed herein include, but are not limited to, chloride, fluoride,acetate, trifluoroacetate, phosphate, sulfate, carbonate, citrate,ascorbate, sorbate, glutarate, ketoglutarate, and combinations thereof.Trifluoroacetate salts of peptide compounds described herein aretypically formed during purification in trifluoroacetic acid buffersusing high-performance liquid chromatography (HPLC). While generally notsuited for in vivo use, trifluoroacetate salt forms of the peptidecompounds described herein may be conveniently used in various in vitrocell culture studies or assays performed to test the activity orefficacy of a peptide compound of interest. The peptide compound maythen be converted from the trifluoroacetate salt (e.g., by ion exchangemethods) to or synthesized as a salt form that is acceptable forpharmaceutical or dietary supplement (nutraceutical) compositions.

A peptide compound useful in the methods of the invention is preferablypurified once it has been isolated or synthesized by either chemical orrecombinant techniques. For purification purposes, there are manystandard methods that may be employed including reversed-phasehigh-pressure liquid chromatography (HPLC) using an alkylated silicacolumn such as C₄-, C₈- or C₁₈-silica. A gradient mobile phase ofincreasing organic content is generally used to achieve purification,for example, acetonitrile in an aqueous buffer, usually containing asmall amount of trifluoroacetic acid. Ion-exchange chromatography canalso be used to separate peptide compounds based on their charge. Thedegree of purity of the peptide compound may be determined by variousmethods, including identification of a major large peak on HPLC. Apeptide compound that produces a single peak that is at least 95% of theinput material on an HPLC column is preferred. Even more preferable is apolypeptide that produces a single peak that is at least 97%, at least98%, at least 99% or even 99.5% of the input material on an HPLC column.

In order to ensure that a peptide compound obtained using any of thetechniques described above is the desired peptide compound for use incompositions and methods of the present invention, analysis of thecompound's composition determined by any of a variety of analyticalmethods known in the art. Such composition analysis may be conductedusing high resolution mass spectrometry to determine the molecularweight of the peptide. Alternatively, the amino acid content of apeptide can be confirmed by hydrolyzing the peptide in aqueous acid, andseparating, identifying and quantifying the components of the mixtureusing HPLC, or an amino acid analyzer. Protein sequenators, whichsequentially degrade the peptide and identify the amino acids in order,may also be used to determine definitely the sequence of the peptide.Since some of the peptide compounds contain amino and/or carboxyterminal capping groups, it may be necessary to remove the capping groupor the capped amino acid residue prior to a sequence analysis.Thin-layer chromatographic methods may also be used to authenticate oneor more constituent groups or residues of a desired peptide compound.

The various peptide compounds described herein are useful in thecompositions and methods of the invention to upregulate the expressionof a gene encoding SOD and/or CAT and thereby generate antioxidativeactivity to counteract the undesirable and destructive oxidativeactivity of ROS and free radicals, e.g., as generated in the agingprocess (senescence), disease, and various drug treatments. Preferredpeptide compounds, excluding any amino and/or carboxy terminal cappinggroup (i.e., the “core sequence”), are less than 20 amino acids inlength. In particular, such preferred peptide compounds, in the absenceof amino and carboxy terminal capping groups, are less than 18, 15, 13,9, 6, 5, 4, and even 3 amino acids in length. A peptide useful in thecompositions and methods of the invention may be 3 or even 2 amino acidsin length (core sequence), such as the preferred dipeptide compound thathas an amino acid sequence consisting of Asp Gly. Any amino terminaland/or carboxy terminal capping group described herein may be added tosuch preferred peptide compounds, provided the capping group does notalso react with other groups in the peptide to result in a significantor toxic amount of undesirable cyclization or polymerization.

The invention provides a peptide compound having the formula:

(SEQ ID NO: 1) R₁ Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln R₂,wherein R₁ is absent or is an amino terminal capping group; R₂ is absentor is a carboxy terminal capping group of the peptide compound; andwherein the peptide compound upregulates expression of a gene encodingan antioxidative enzyme.

In another embodiment, the invention provides a peptide compound havingthe formula:

R₁ Gln Thr Leu Gln Phe Arg R₂, (SEQ ID NO: 2)wherein R₁ is absent or is an amino terminal capping group; R₂ is absentor is a carboxy terminal capping group of the peptide compound; andwherein the peptide compound upregulates expression of a gene encodingan antioxidative enzyme.

In yet another embodiment, the invention provides a peptide compoundhaving the formula:R₁ Xaa₁ Gly Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ R2  (SEQ ID NO:3),wherein Xaa₁ and Xaa₂ are, independently, aspartic acid or asparagine;R₁ is absent or is an amino terminal capping group of the peptidecompound; Xaa₃ is absent or Gly; Xaa₄ is absent, Asp, or Phe; Xaa₅ isabsent, Ala, or Phe; Xaa₆ is absent or Ala; R₂ is absent or is a carboxyterminal capping group of the peptide compound; and wherein the peptidecompound upregulates expression of a gene encoding an antioxidativeenzyme. A preferred peptide compound according to the formula,upregulates expression of a gene encoding an antioxidative enzyme andcomprises an amino acid sequence selected from the group consisting of:

Asp Gly Asp Asp Gly Asn Asn Gly Asn Asn Gly Asp Asp Gly Asp Gly Asp,(SEQ ID NO: 4) Asp Gly Asp Gly Phe Ala, (SEQ ID NO: 5)Asp Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 6) Asp Gly Asn Gly Asp Phe Ala,(SEQ ID NO: 7) Asn Gly Asn Gly Asp Phe Ala, (SEQ ID NO: 8) andAsn Gly Asp Gly Asp Phe Ala. (SEQ ID NO: 9)

The invention also provides a peptide compound having the formula:R₁ Asn Ser Thr R₂,wherein R₁ is absent or is an amino terminal capping group; R₂ is absentor is a carboxy terminal capping group of the peptide compound; andwherein the peptide compound upregulates expression of a gene encodingan antioxidative enzyme.

In still another embodiment, the invention provides a peptide compoundhaving the formula:R₁ Phe Asp Gln R₂,wherein R₁ is absent or is an amino terminal capping group; R₂ is absentor is a carboxy terminal capping group of the peptide compound; andwherein the peptide compound upregulates expression of a gene encodingan antioxidative enzyme.

The invention also provides a peptide compound having the formula:

(SEQ ID NO: 10) R₁ Xaa₁ Xaa₂ Met Thr Leu Thr Gln Pro R₂,wherein Xaa₁ is absent or Ser; Xaa₂ is absent or Lys; R₁ is absent or anamino terminal capping group; R₂ is absent or a carboxy terminal cappinggroup of the peptide compound; and wherein the peptide compoundupregulates expression of a gene encoding an antioxidative enzyme. Apreferred peptide compound according to the formula upregulatesexpression of a gene encoding an antioxidative enzyme and comprises anamino acid sequence selected from the group consisting of

Met Thr Leu Thr Gln Pro (SEQ ID NO: 11) andSer Lys Met Thr Leu Thr Gln Pro. (SEQ ID NO: 12)

Another aspect of the invention is a peptide compound having theformula:R₁ Xaa₁ Xaa₂ Xaa₃ R₂,wherein Xaa₁ is Asp, Asn, Glu, Gln, Thr, or Tyr, Xaa₂ is absent or anyamino acid (i.e., is variable); Xaa₃ is Asp, Asn, Glu, Thr, Ser, Gly, orLeu; R₁ is absent or is an amino terminal capping group and R₂ is absentor is a carboxy terminal capping group of the peptide compound; whereinthe peptide compound upregulates expression of a gene encoding anantioxidative enzyme. Preferably, a peptide compound of the formulaupregulates expression of a gene encoding an antioxidative enzyme andcomprises the above formula wherein Xaa₂ is selected from the groupconsisting of Val, Gly, Glu, and Gln. More preferably, the peptidecompound of the formula upregulates expression of a gene encoding anantioxidative enzyme and is selected from the group consisting of:

Asp Gly, Asn Gly, Glu Gly, Gln Gly, Thr Val Ser, Asp Gly Asp, and AsnGly Asn.

In still another embodiment, the invention provides a peptide compoundhaving the formula:R₁ Leu Xaa₁ Xaa₂ R₂,wherein Xaa₁ is any amino acid; Xaa₂ is Gln, Gly, or Tyr; R₁ is absentor is an amino terminal capping group; R₂ is absent or is a carboxyterminal capping group of the peptide; and wherein the peptide compoundupregulates expression of a gene encoding an antioxidative enzymecompound.

The invention also provides a peptide compound having the formula:R₁ Met Thr Xaa₁ R₂,wherein Xaa₁ is Asn, Asp, Glu, Thr, or Leu; R₁ is absent or is an aminoterminal capping group; R₂ is absent or is a carboxy terminal cappinggroup of the peptide compound; and wherein the peptide compoundupregulates expression of a gene encoding an antioxidative enzyme.Preferably, the peptide compound of the formula upregulates expressionof a gene encoding an antioxidative enzyme and comprising an amino acidsequence selected from the group consisting of: Met Thr Leu; Met ThrAsp; Met Thr Asn; Met Thr Thr; Met Thr Glu; and Met Thr Gln.

In a preferred embodiment, a peptide compound of any of the formulasdescribed herein has the R₁ amino terminal capping group. Morepreferably, the R₁ amino terminal capping group is selected from thegroup consisting of a lipoic acid moiety (Lip, in reduced or oxidizedform); a glucose-3-O-glycolic acid moiety (Gga); 1 to 6 lysine residues;1 to 6 arginine residues; an acyl group of the formula R₃—CO—, where COis a carbonyl group, and R₃ is a hydrocarbon chain having from 1 to 25carbon atoms, and preferably 1 to 22 carbon atoms, and where thehydrocarbon chain may be saturated or unsaturated and branched orunbranched; and combinations thereof. More preferably, when the aminoterminal capping group is an acyl group it is acetyl or a fatty acid.Even more preferably, the amino terminal capping group is an acyl groupselected from the group consisting of acetyl (Ac), palmitic acid (i.e.,a palmitoyl group, Palm), and docosahexaenoic acid (DHA). In anotherembodiment, the amino terminal capping group is a peptide consisting ofany combination of arginine and lysine wherein the peptide is not lessthan two amino acids in length and not more than six amino acids inlength.

Particularly preferred peptide compounds that upregulate a gene encodingan antioxidative enzyme and that are useful in compositions and methodsof the invention include, but are not limited to, peptide compoundscomprising an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 1) Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln,(SEQ ID NO: 2) Gln Thr Leu Gln Phe Arg, (SEQ ID NO: 13)Glu Thr Leu Gln Phe Arg, (SEQ ID NO: 14)Gln Tyr Ser Ile Gly Gly Pro Gln, (SEQ ID NO: 15)Ser Asp Arg Ser Ala Arg Ser Tyr, (SEQ ID NO: 12)Ser Lys Met Thr Leu Thr Gln Pro, (SEQ ID NO: 13)Met Thr Leu Thr Gln Pro, (SEQ ID NO: 16)Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu, (SEQ ID NO: 6)Asp Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 4) Asp Gly Asp Gly Asp,(SEQ ID NO: 8) Asn Gly Asn Gly Asp Phe Ala, (SEQ ID NO: 17)Asn Gly Asn Gly Asp (SEQ ID NO: 7) Asp Gly Asn Gly Asp Phe Ala,(SEQ ID NO: 18) Asp Gly Asn Gly Asp, (SEQ ID NO: 9)Asn Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 19) Asn Gly Asp Gly Asp,(SEQ ID NO: 20) Asn Gly Asp Gly, (SEQ ID NO: 5) Asp Gly Asp Gly Phe Ala,(SEQ ID NO: 21) Asn Gly Asn Gly Phe Ala, (SEQ ID NO: 22)Asp Gly Asn Gly Phe Ala, (SEQ ID NO: 23) Asn Gly Asp Gly Phe Ala,Asp Gly Asp, Asn Gly Asn, Asp Gly Asn, Asn GlyAsp, Asn Ser Thr, Phe Asp Gln, Met Thr Leu, MetThr Asp, Met Thr Asn, Met Thr Thr, Met Thr Glu,Met Thr Gln, Thr Val Ser, Leu Thr Gln, Leu ThrGly, Leu Thr Tyr, Asp Gly, Asn Gly, Glu Gly, GlnGly, Glu Ala, Gln Ala, Gln Gly, Asp Ala, and Asn Ala.

A particularly preferred peptide compound of the invention thatupregulates a gene encoding an antioxidative enzyme and that is usefulin compositions and methods of the invention comprises an amino acidsequence selected from the group consisting of: Asp Gly Asp, Thr ValSer, Asp Gly, and Glu Ala.

Such preferred peptide compounds as listed above may also contain one ormore terminal capping groups, such as an amino terminal capping groupand/or a carboxy terminal capping group described herein. Preferredpeptide compounds containing one or more terminal capping groups thatupregulate an antioxidative enzyme and that are useful in thecompositions and methods include, but are not limited to, peptidecompounds having the formulas:

(SEQ ID NO: 25) Lys Lys Glu Thr Leu Gln Phe Arg; (SEQ ID NO: 26)Lys Lys Gln Thr Leu Gln Phe Arg; (SEQ ID NO: 27)Lys Lys Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu; (SEQ ID NO: 27)DHA Lys Lys Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu;(SEQ ID NO: 11) Palm Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu;(SEQ ID NO: 12) Gga Asp Gly Asp Gly Asp Phe Ala; (SEQ ID NO: 12)Ac Asp Gly Asp Gly Asp Phe Ala; (SEQ ID NO: 12)Palm Asp Gly Asp Gly Asp Phe Ala; (SEQ ID NO: 13)Gga Asp Gly Asp Gly Asp; (SEQ ID NO: 13) Palm Asp Gly Asp Gly Asp;(SEQ ID NO: 13) Lip Asp Gly Asp Gly Asp; (SEQ ID NO: 13)DHA Asp Gly Asp Gly Asp; (SEQ ID NO: 13) (Lys)_(n) Asp Gly Asp Gly Asp;Ac-Thr Val Ser; Lip Thr Val Ser; Gga Asp Gly Asp;Palm Asp Gly Asp; Lip Asp Gly Asp; DHA Asp GlyAsp; (Lys)_(n) Asp Gly Asp; Gga Phe Asp Gln; Palm PheAsp Gln; Lip Phe Asp Gln; DHA Phe Asp Gln (Lys)_(n)Phe Asp Gln; Gga Asn Ser Thr; Palm Asn Ser Thr;Lip Asn Ser Thr; DHA Asn Ser Thr; (Lys)_(n) Asn SerThr; Gga Asp Gly; Palm Asp Gly Lip Asp Gly; DHAAsp Gly; (Lys)_(n) Asp Gly; Gga Asn Gly; Palm AsnGly; Lip Asn Gly; DHA Asn Gly; (Lys)_(n) Asn Gly; GgaGlu Ala; Palm Glu Ala; DHA Glu Ala; (Lys)_(n) GluAla; Gga Gln Ala; Palm Gln Ala; DHA Gln Ala;(Lys)_(n) Gln Ala; Gga Glu Gly; Palm Glu Gly; DHA GluGly; (Lys)_(n) Glu Gly; Gga Glu Gly; Palm Glu Gly;DHA Glu Gly; (Lys)_(n) Glu Gly; Gga Gln Gly; Palm GlnGly; DHA Gln Gly; and (Lys)_(n) Gln Gly,wherein Palm is a palmitic acid (palmitoyl) group, Lip is lipoic acidgroup, in either oxidized or reduced form; Ac is an acetyl group; DHA isan docosahexaenoic acid group; Gga is a glucose-3-O-glycolic acid group;and n in (Lys)_(n) is 1-6. These preferred peptide compounds may alsocontain a carboxy terminal capping group, such as a primary amino groupin amide linkage to the carboxy terminal amino acid.

One aspect of the invention contemplates a metabolically convertibleform of an peptide compound of the invention wherein asparagine andglutamine residues in the amino acid sequence of the peptide compoundsare converted in a cell to their corresponding acid form, or saltthereof in physiological conditions, i.e., aspartic acid (or aspartate)and glutamic acid (or glutamate). For example, peptides consisting ofthe amino acid sequences Asn Gly and Gln Gly are contemplated to bemetabolically converted to the corresponding peptides consisting of AspGly and Glu Gly, respectively, upon administration and uptake by cells.Accordingly, a peptide compound useful in a composition or method of theinvention that comprises an amino acid sequence comprising one or moreasparagine and/or glutamine residues also provides a disclosure of acorresponding peptide compound in which aspartate and/or glutamate aresubstituted for asparagine and/or glutamine residues, respectively, andvice versa.

Biological and Biochemical Activities

The peptide compounds useful in the compositions and methods of theinvention have the ability to upregulate SOD and/or CAT, as well asactivate and upregulate AP-1 in cells and tissues, especially mammaliancells, provided the cells contain at least one functional gene encodinga SOD or CAT protein. A functional gene is one, which not only encodesthe particular enzyme, but also provides the necessary geneticinformation within and without the coding sequence so that transcriptionof the gene can occur and so that the mRNA transcript can be translatedinto a functioning gene product.

Certain preferred peptide compounds described herein are able toupregulate both SOD and CAT, assuming that functional genes for bothenzymes are present in the cells of interest. Advantageously,upregulation of SOD and CAT together provide enhanced efficacy indetoxifying undesired ROS and free radicals. Without wishing to be boundby theory, when the level of SOD protein increases as a result of SODupregulation, it is believed that superior antioxidative efficacy isachieved when there is also an increase in CAT levels. Upregulation of agene for CAT increases the capacity to neutralize and detoxify theadditional hydrogen peroxide and other ROS or free radicals that can begenerated by enhanced SOD activity. The peptide compounds describedherein having both SOD and CAT upregulation activity provide cells andtissues with a full complement of enhanced antioxidative enzyme activityto detoxify ROS and free radicals. For example, contacting mammaliancells in tissue culture with a peptide compound described herein havingboth SOD and CAT upregulation activity typically results in at leastabout a 2-fold (and in increasing order of preference, at least about a3-fold, 4-fold, and a 6 to 8-fold) increase in the levels of SOD and CATmRNA transcripts and about a 2-fold (and in increasing order ofpreference, at least about a 3-fold, 4-fold, 6-fold, 8-fold, 10-fold anda 12- to 14-fold) increase in the levels of SOD and CAT protein, asdetected by immunoblotting and compared to untreated cells. Suchincrease in SOD and CAT gene expression levels provides a cell with asignificantly enhanced capability for detoxifying ROS and free radicalswithout adverse effects.

Expression of genes encoding SOD, CAT, and AP-1 can be measured by avariety of methods. Standard enzymatic assays are available to detectlevels of SOD and CAT in cell and tissue extracts or biological fluids(Fridovich, Adv. Enzymol., 41:35-97 (1974); Beyer & Fridovich, Anal.Biochem., 161:559-566 (1987)). In addition, antibodies to SOD, CAT, andAP-1 are available or readily made. Using such antibodies specific foreach protein, standard immunoblots (e.g., Western blots) and otherimmunological techniques can be used to measure levels of SOD and CAT invarious mixtures, cell extracts, or other sample of biological material.Provided there is no evidence of a defect in the translation machineryof the cells of interest, the levels of expression of genes encodingSOD, CAT, and AP-1 can also be measured by detecting levels of mRNAtranscripts using standard Northern blot or standard polymerase chainreaction (PCR) methods for measuring specific mRNA species (e.g.,RT-PCR). In addition, activation and translocation of AP-1 to nuclei canbe determined using a standard electrophoretic mobility shift assay(EMSA) in which the amount of AP-1 protein present in cell nuclei isdetected by its ability to form a complex with a DNA probe moleculecontaining a specific DNA sequence for a promoter/enhancer region of aeukaryotic gene, which is known to be bound by AP-1. Typically, the AP-1protein-DNA complex migrates with a slower mobility than unbound DNA.The AP-1 transcription factor complex is formed by the association ofother factors, such as c-Jun and c-Fos, with a DNA molecule containingan AP-1 recognition sequence or site. The presence of AP-1 in a mixtureor sample is then detected by the formation of such protein-DNAmolecular complexes, which result in an observable shift in theelectrophoretic mobility from the position of the uncomplexed DNA in agel.

Other preferred peptide compounds useful in the compositions and methodsof the invention are able to upregulate levels of the AP-1 transcriptionfactor. For example, contacting mammalian cells in tissue culture withpeptide compounds described herein typically results in at least about a2-fold and, in order of increasing preference, at least about a 6-fold,8-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, and 20- to 60-foldincrease in the level of AP-1 protein, as determined by EMSA. Suchupregulation of AP-1 gene expression can result in an enhanced AP-1dependent gene expression.

Therapeutic and Prophylactic Applications

The peptide compounds of the invention upregulate SOD and/or CAT incells and tissues of animals, such as humans and other mammals.Preferably, the peptides of this invention upregulate both SOD and CAT.As noted above, SOD and CAT comprise components of the body's majorenzymatic antioxidative activities that are able to detoxify ROS andfree radicals by reducing such molecules to less reactive and lessharmful compounds. The contribution of ROS and other free radicals tothe progression of various disease states and side effects of drugs isnow well known.

For example, elevated levels of ROS and free radicals are known to begenerated in cells and tissues during reperfusion after an ischemicevent. Such increased levels of ROS and free radicals can causeconsiderable damage to an already stressed or debilitated organ ortissue. The peptide compounds of this invention, which upregulate SODand/or CAT, may be used to treat reperfusion injuries that occur indiseases and conditions such as stroke, heart attack, or renal diseaseand kidney transplants. If the ischemic event has already occurred as instroke and heart attack, a peptide compound described herein may beadministered to the individual to detoxify the elevated ROS and freeradicals already present in the blood and affected tissue or organ.Alternatively, if the ischemic event is anticipated as in organtransplantation, then peptide compounds described herein may beadministered prophylactically, prior to the operation or ischemic event.

Although a major application is in the treatment of ischemia-reperfusioninjury, the peptide compounds described herein may be used to treat anydisease or condition associated with undesirable levels of ROS and freeradicals or to prevent any disease, disorder or condition caused byundesirable levels of ROS and free radicals. According to the invention,the peptide compounds described herein may also be administered toprovide a therapeutic or prophylactic treatment of elevated ROS andother free radicals associated with a variety of other diseases andconditions, including, but not limited to, oxygen toxicity in prematureinfants, burns and physical trauma to tissues and organs, septic shock,polytraumatous shock, head trauma, brain trauma, spinal cord injuries,Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer'sdisease, age-related elevation of ROS and free radicals, senility,ulcerative colitis, human leukemia and other cancers, Down syndrome,arthritis, macular degeneration, schizophrenia, epilepsy, radiationdamage (including UV-induced skin damage), and drug-induced increase inROS and free radicals.

A progressive rise of oxidative stress due to the formation of ROS andfree radicals occurs during aging (see, e.g., Mecocci, P. et al., FreeRadic. Biol. Med., 28: 1243-1248 (2000)). This has been detected byfinding an increase in the formation of lipid peroxidates in rat tissues(Erdincler, D. S., et al., Clin. Chim. Acta, 265: 77-84 (1997)) andblood cells in elderly human patients (Congi, F., et al., Presse. Med.,24: 1115-1118 (1995)). A recent review (Niki, E., Intern. Med., 39:324-326 (2000)) reported that increased tissue damage by ROS and freeradicals could be attributed to decreased levels of the antioxidativeenzymes SOD and CAT that occurs during aging. For example, transgenicanimals, generated by inserting extra SOD genes into the genome of micewere found to have decreased levels of ROS and free radical damage. Suchanimals also had an extended life span. More recent evidence indicatedthat administration of a small manganese porphyrin compound, whichmimics SOD activity, led to a 44% extension of life span of the nematodeworm Caenorhabditis elegans (S. Melow, et al., Science, 289: 1567-1569(2000)). Accordingly, the peptide compounds described herein, which areable to upregulate expression of SOD and/or CAT genes to produceincreased levels of antioxidative enzymes, are also well suited for usein methods of preventing and/or counteracting increased tissue damageand decreased life expectancy due to elevated levels of ROS and freeradicals that accompany the aging process.

A variety of drugs in current therapeutic use produce tissue-specifictoxic side effects that are correlated with an elevation in the levelsof ROS and other free radicals. Such drugs include neuroleptics,antibiotics, analgesics, and other classes of drugs. The tissuesaffected by such drug-induced toxicities can include one or more of themajor organs and tissues, such as brain, heart, lungs, liver, kidney,and blood.

One of the most dangerous side effects of a drug has been reported forthe neuroleptic, clozapine, which was the first drug with majorpotential as an anti-schizophrenic therapeutic activity (see, Somani etal., In Oxidants, Antioxidants And Free Radicals (S. I. Baskin And H.Salem, eds.) (Taylor And Francis, Washington D.C., 1997), pages125-136). Approximately 1-2% of clozapine-treated patients developagranulocytosis, which is correlated with the production of ROS Fischeret al., Molecular Pharm., 40:846-853, 1991). According to the invention,a peptide compound as described herein is administered toclozapine-treated patients to upregulate the SOD and/or CAT, whichcounteracts the undesirable and harmful increase in ROS and other freeradicals and, thereby, reduces the risk of developing agranulocytosis.

Another side effect of schizophrenic patients receiving neuroleptics isTardive dyskinesia, which is a debilitating disease manifested byvarious uncontrollable oral, facial, and/or trunk movements. Manypatients, especially veterans in hospital, suffer permanent disabilityfrom this unfortunate, drug-induced disease. Previous studies on Tardivedyskinesia were focused on the loss of dopamine neurons (see, forexample, Morganstern and Glazer, Arch. Gen. Psychiatr., 50: 723-733(1993)). However, more recent studies have demonstrated that the primarydefect in brains of such patients is the overproduction of theexcitotoxic amino acid glutamate in the presynaptic input to thestriatal dopaminergic neurons. Notably, this overproduction of glutamateproduces excitotoxic effects on dopamine cells by causing a highincrease in ROS and free radicals (see, Tsai et al., Am. J. Psychiatr.,155: 1207-1253 (1998)). Accordingly, the peptide compounds of thisinvention may be administered to patients receiving neuroleptics toupregulate SOD and/or CAT and thereby provide the enhanced antioxidativeactivities to counteract the oxidative effects of the elevated levels ofROS and free radicals.

According to the methods of the invention, peptide compounds describedherein may be administered to an individual before, contemporaneouslywith, or after administration of a therapeutic drug whose use has beencorrelated with the undesirable side effect of elevation in the levelsof ROS and other free radicals. Such drugs include, but are not limitedto those listed in Table 1 (see, Somani et al., 1997), which also listsany known manifested toxicity or side effect.

TABLE 1 ROS or Reactive Free Radical DRUG or Toxic Result Toxicityclozapine ROS and free radicals agranulocytosis doxorubin superoxideanion, cardiac (anthrcyclines) hydroxyl radical bleomycin superoxideanion pulmonary mytomycin free radical cisplatin probably free radicalnephrotoxicity, otototoxicity BCNU (carmustine) methyl radicalneurotoxicity procarbazine free radical neurotoxicity acetaminophenreactive intermediate metabolites hepatic of drug isoniazid free radicalhepatic ethanol α-hydroxy ethyl radical hepatic, neurotoxicityphysostigmine eseroline to catechol to quinones neurotoxicity quinonesreactive metabolites of drug neurotoxicity morphine covalent bindingreactions neurotoxicity nitrofurantoin oxidant pulmonary paraquatoxidant pulmonary parathion reactive metabolites of drug neruotoxicitycarbon tetrachloride trichloromethyl radical hepatic (CCl₄) polycyclicaromatic reactive epoxides hepatic hydrocarbons nitrofurazone ROS andfree radicals pulmonary metronidazole ROS and free radicals pulmonary6-hydroxydopamine ROS and free radicals neurotoxin 4-hydroxyanisole freeradicals etoposide (VP-16) hydroxyl radicals benzidine free radicalsbladder carcinogen aminopyrine free radicals agranulocytosis clozarilfree radicals agranulocytosis phenylhydrazine ROS and free radicalshemolytic anemia 3-methylindole free radicals pulmonary probucol freeradicals ferrous sulfate hydroxyl radicals iron overload methimazolefree radicals chloroprazine free radicals phototoxicity, photoallergysalicylanilides free radicals photoallergy mitoxantrone free radicalsdaunomycin ROS and free radicals cardiotoxicityPharmaceutical Applications

Pharmaceutical compositions of this invention comprise any of thepeptide compounds of the present invention, and pharmaceuticallyacceptable salts thereof, with any pharmaceutically acceptableingredient, excipient, carrier, adjuvant or vehicle.

Pharmaceutical compositions of this invention can be administered tomammals, including humans, in a manner similar to other therapeutic,prophylactic, or diagnostic agents, and especially therapeutic hormonepeptides. The dosage to be administered, and the mode of administrationwill depend on a variety of factors including age, weight, sex,condition of the patient, and genetic factors, and will ultimately bedecided by the attending physician or veterinarian. In general, dosagerequired for diagnostic sensitivity or therapeutic efficacy will rangefrom about 0.001 to 25.0 μg/kg of host body mass.

Pharmaceutically acceptable salts of the peptide compounds of thisinvention include, for example, those derived from pharmaceuticallyacceptable inorganic and organic acids and bases. Examples of suitableacids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, malic, pamoic, phosphoric, glycolic, lactic, salicylic,succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,formic, benzoic, malonic, naphthalene-2-sulfonic, tannic, carboxymethylcellulose, polylactic, polyglycolic, and benzenesulfonic acids. Otheracids, such as oxalic, while not in themselves pharmaceuticallyacceptable, may be employed in the preparation of salts useful asintermediates in obtaining the compounds of the invention and theirpharmaceutically acceptable acid addition salts. Salts derived fromappropriate bases include alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N—(C₁₋₄ alkyl)₄ ⁺ salts.

This invention also envisions the “quaternization” of any basicnitrogen-containing groups of a peptide compound disclosed herein,provided such quaternization does not destroy the ability of the peptidecompound to upregulate expression of genes encoding SOD and CAT, and,where desired, AP-1. Such quaternization may be especially desirablewhere the goal is to use a peptide compound containing only positivelycharged residues. As noted above, in a most preferred embodiment of theinvention, when charged amino acid residues are present in a peptidecompound described herein, they are either all basic (positivelycharged) or all acidic (negatively) which prevents formation of cyclicpeptide compounds during storage or use. Typically, cyclic forms of thepeptide compounds are inactive and potentially toxic. Thus, aquaternized peptide compound is a preferred form of a peptide compoundcontaining basic amino acids. Even more preferred is the quaternizedpeptide compound in which the carboxy terminal carboxyl grouped isconverted to an amide to prevent the carboxyl group from reacting withany free amino groups to form a cyclic compound. Any basic nitrogen canbe quaternized with any agent known to those of ordinary skill in theart including, for example, lower alkyl halides, such as methyl, ethyl,propyl and butyl chloride, bromides and iodides; dialkyl sulfatesincluding dimethyl, diethyl, dibutyl and diamyl sulfates; long chainhalides such as decyl, lauryl, myristyl and stearyl chlorides, bromidesand iodides; and aralkyl halides including benzyl and phenethylbromides. Water or oil-soluble or dispersible products may be obtainedby such quaternization or acids such as acetic acid and hydrochloricacid.

It should be understood that the peptide compounds of this invention maybe modified by appropriate functionalities to enhance selectivebiological properties, and in particular the ability to upregulate SODand/or CAT and/or AP-1. Such modifications are known in the art andinclude those, which increase the ability of the peptide compound topenetrate or being transported into a given biological system (e.g.,brain, central nervous system, blood, lymphatic system), increase oralavailability, increase solubility to allow administration by injection,alter metabolism of the peptide compound, and alter the rate ofexcretion of the peptide compound. In addition, the peptide compoundsmay be altered to a pro-drug form such that the desired peptide compoundis created in the body of the patient as the result of the action ofmetabolic or other biochemical processes on the pro-drug. Such pro-drugforms typically demonstrate little or no activity in in vitro assays.Some examples of pro-drug forms may include ketal, acetal, oxime, andhydrazone forms of compounds which contain ketone or aldehyde groups.Other examples of pro-drug forms include the hemi-ketal, hemi-acetal,acyloxy ketal, acyloxy acetal, ketal, and acetal forms.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The pharmaceutical compositions of this invention may be administered bya variety of routes or modes. These include, but are not limited to,parenteral, oral, intratracheal, sublingual, pulmonary, topical, rectal,nasal, buccal, sublingual, vaginal, or via an implanted reservoir.Implanted reservoirs may function by mechanical, osmotic, or othermeans. The term “parenteral”, as understood and used herein, includesintravenous, intracranial, intraperitoneal, paravertebral,periarticular, periostal, subcutaneous, intracutaneous, intra-arterial,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, and intralesional injection or infusion techniques. Suchcompositions are preferably formulated for parenteral administration,and most preferably for intravenous, intracranial, or intra-arterialadministration. Generally, and particularly when administration isintravenous or intra-arterial, pharmaceutical compositions may be givenas a bolus, as two or more doses separated in time, or as a constant ornon-linear flow infusion.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as those described in Pharmacoplia Halselica.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, caplets, pills, aqueous or oleaginoussuspensions and solutions, syrups, or elixirs. In the case of tabletsfor oral use, carriers, which are commonly used include lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. Capsules, tablets, pills,and caplets may be formulated for delayed or sustained release.

When aqueous suspensions are to be administered orally, the peptidecompound is advantageously combined with emulsifying and/or suspendingagents. If desired, certain sweetening and/or flavoring and/or coloringagents may be added. Formulations for oral administration may contain10%-95% active ingredient, preferably 25%-70%. Preferably, apharmaceutical composition for oral administration provides a peptidecompound of the invention in a mixture that prevents or inhibitshydrolysis of the peptide compound by the digestive system, but allowsabsorption into the blood stream.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for vaginal or rectaladministration. These compositions can be prepared by mixing a compoundof this invention with a suitable non-irritating excipient, which issolid at room temperature but liquid at body temperature and thereforewill melt in relevant body space to release the active components. Suchmaterials include, but are not limited to, cocoa butter, beeswax andpolyethylene glycols. Formulations for administration by suppository maycontain 0.5%-10% active ingredient, preferably 1%-2%.

Topical administration of the pharmaceutical compositions of thisinvention may be useful when the desired treatment involves areas ororgans accessible by topical application, such as in wounds or duringsurgery. For application topically, the pharmaceutical composition maybe formulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the peptide compounds of this invention include, but are not limitedto, mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the peptide compounds suspended ordissolved in a pharmaceutically suitable carrier. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water. The pharmaceutical composition may beformulated for topical or other application as a jelly, gel, oremollient, where appropriate. The pharmaceutical compositions of thisinvention may also be topically applied to the lower intestinal tract byrectal suppository formulation or in a suitable enema formulation.Topical administration may also be accomplished via transdermal patches.This may be useful for maintaining a healthy skin tissue and restoringoxidative skin damage (e.g., UV- or radiation-induced skin damage).

The pharmaceutical compositions of this invention may be administerednasally, in which case absorption may occur via the mucus membranes ofthe nose, or inhalation into the lungs. Such modes of administrationtypically require that the composition be provided in the form of apowder, solution, or liquid suspension, which is then mixed with a gas(e.g., air, oxygen, nitrogen, etc., or combinations thereof) so as togenerate an aerosol or suspension of droplets or particles. Suchcompositions are prepared according to techniques well-known in the artof pharmaceutical formulation and may be prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, fluorocarbons, and/orother solubilizing or dispersing agents known in the art.

Pharmaceutical compositions of the invention may be packaged in avariety of ways appropriate to the dosage form and mode ofadministration. These include but are not limited to vials, bottles,cans, packets, ampoules, cartons, flexible containers, inhalers, andnebulizers. Such compositions may be packaged for single or multipleadministrations from the same container. Kits, of one or more doses, maybe provided containing both the composition in dry powder or lyophilizedform, as well an appropriate diluent, which are to be combined shortlybefore administration. The pharmaceutical composition may also bepackaged in single use pre-filled syringes, or in cartridges forauto-injectors and needleless jet injectors.

Multi-use packaging may require the addition of antimicrobial agentssuch as phenol, benzyl alcohol, meta-cresol, methyl paraben, propylparaben, benzalconium chloride, and benzethonium chloride, atconcentrations that will prevent the growth of bacteria, fungi, and thelike, but be non-toxic when administered to a patient.

Consistent with good manufacturing practices, which are in current usein the pharmaceutical industry and which are well known to the skilledpractitioner, all components contacting or comprising the pharmaceuticalagent must be sterile and periodically tested for sterility inaccordance with industry norms. Methods for sterilization includeultrafiltration, autoclaving, dry and wet heating, exposure to gasessuch as ethylene oxide, exposure to liquids, such as oxidizing agents,including sodium hypochlorite (bleach), exposure to high energyelectromagnetic radiation, such as ultraviolet light, x-rays or gammarays, and exposure to ionizing radiation. Choice of method ofsterilization will be made by the skilled practitioner with the goal ofeffecting the most efficient sterilization that does not significantlyalter a desired biological function, i.e., the ability to upregulateSOD, CAT, or AP-1, of the pharmaceutical agent in question.Ultrafiltration is a preferred method of sterilization forpharmaceutical compositions that are aqueous solutions or suspensions.

Details concerning dosages, dosage forms, modes of administration,composition and the like are further discussed in a standardpharmaceutical text, such as Remington's Pharmaceutical Sciences, 18thed., Alfonso R Gennaro, ed. (Mack Publishing Co., Easton, Pa. 1990),which is hereby incorporated by reference.

As is well known in the art, structure and biological function ofpeptides are sensitive to chemical and physical environmental conditionssuch as temperature, pH, oxidizing and reducing agents, freezing,shaking and shear stress. Due to this inherent susceptibility todegradation, it is necessary to ensure that the biological activity of apeptide compound used as a pharmaceutical agent be preserved during thetime that the agent is manufactured, packaged, distributed, stored,prepared and administered by a competent practitioner. Many technicalapproaches have been developed to stabilize pharmaceutical proteins soas to preserve their biological potency and efficacy, and suchstabilizing techniques may be applied to peptide compounds of thecompositions and methods of the invention, including:

a) Freeze-drying and lyophilization (refer to Carpenter et al., Pharm.Res., 14(8): 969 (1997), incorporated by reference);

b) Addition of “stabilizers” to the aqueous solution or suspension ofthe peptide or protein. For example, U.S. Pat. No. 5,096,885 disclosesaddition of glycine, mannitol, pH buffers, and the non-ionic surfactantpolysorbate 80 to human growth hormone as means to stabilize the proteinduring the process of filtration, vial filling, and cold storage orlyophilization; U.S. Pat. No. 4,297,344 discloses stabilization ofcoagulation factors II and VIII, antithrombin III and plasminogenagainst heat by adding selected amino acids and a carbohydrate; U.S.Pat. No. 4,783,441 discloses a method for prevention of denaturation ofproteins such as insulin in aqueous solution at interfaces by theaddition of surface acting substances, within a particular pH range; andU.S. Pat. No. 4,812,557 discloses a method of stabilizing interleukin-2using human serum albumin;

c) Freeze/thaw methods wherein the peptide compound is mixed with acryoprotectant and stored frozen at very low temperatures (e.g., −70°C.);

d) Cold, non-frozen storage (e.g., less than 4° C.), optionally with acryoprotectant additive such as glycerol;

e) Storage in a vitrified, amorphous state, e.g., as described in U.S.Pat. No. 5,098,893;

f) Storage in a crystalline state; and

g) Incorporation into liposomes or other micelles.

Natural Source, Purified Compositions and Dietary Supplements

The invention also provides compositions and methods of making suchcompositions for use as dietary supplements (also referred to as“nutraceuticals”) comprising a natural source purified compositionobtained from an organism (i.e., animal, plant, or microorganism), whichpurified composition contains an endogenous peptide compound describedherein, which upregulates expression of one or more genes encoding anantioxidative enzyme, such as SOD and/or CAT in cells or tissues.Although peptide compounds of the invention may be obtained in highlypurified form from some natural sources, the level of such peptidecompounds in natural materials may be quite low or even present in onlya trace detectable amount, even in compositions purified from suchnatural sources. Accordingly, to obtain useful quantities of purepeptide compounds, it is usually more economical to synthesize thepeptide compounds described herein using in vitro automated peptidesynthesis protocols. However, purified preparations from natural sourcesthat contain even trace amounts of a compound capable of upregulating anantioxidative enzyme (such as SOD and/or CAT) may be useful inmanufacturing products for sale in the oral dietary supplements market.Accordingly, dietary supplement compositions of the invention mayfurther comprise an exogenously provided peptide compound describedherein that upregulates expression of one or more genes encoding anantioxidative enzyme, such as SOD and/or CAT. Preferred natural sourcesof purified compositions used in making dietary supplements of theinvention include green velvet antler from a ruminant, such as deer orelk, and various plant tissue, such as roots, stems, leaves, flowers,herbal mixtures, and teas. A preferred natural plant source useful inpreparing dietary supplements of the invention is wuzi yanzong herbalmixture. Wuzi yanzong herbal mixture has been reported to confer onindividuals a number of beneficial effects, including elevation oflevels of certain antioxidative enzymes such as SOD and bloodglutathione peroxidase, that make it a desirable natural source for usein manufacturing marketable dietary supplements of the invention (see,e.g., abstracts from Huang et al., Chung Kuo Chung Yao Tsa Chih, 16:414-416, 447 (1991); Wang et al., Chung Kuo Chung Hsi I Chieh Ho TsaChih, 12: 23-25, 5 (1992); Wang et al., Chung Kuo Chung Hsi I Chieh HoTsa Chih, 13: 349-351, 325-326 (1993); Li et al., Chung Kuo Chung YaoTsa Chih, 19: 300-302 (1994)).

Dietary supplement formulations of the invention may comprise a naturalsource purified composition comprising an endogenous peptide compounddescribed herein. Other dietary supplement formulations of the inventionare compositions which comprise a natural source purified compositioncontaining an endogenous peptide compound, which is combined with one ormore exogenously provided peptide compounds described herein. Anadvantage of this latter type of formulation is that a sufficient amountof an exogenously provided peptide compound described herein may becombined with the natural source purified composition to form a dietarysupplement composition that produces a desirable level or range oflevels of upregulated antioxidative enzymes in an individual that takesor is administered the dietary supplement. Accordingly, dietarysupplement compositions of the invention may contain one or moredifferent peptide compounds described herein as an endogenous peptidecompound from a natural source purified composition as well as, if soformulated, an exogenously provided peptide compound described herein.According to the invention, dietary supplements may comprise anendogenous peptide compound and an exogenously provided peptide compoundthat are the same or different peptide compounds.

Preferred dietary supplements of the invention comprise a peptidecompound comprising the formulas:R₁ Xaa₁ Gly Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ R₂  (SEQ ID NO:3),

wherein Xaa₁ and Xaa₂ are, independently, aspartic acid or asparagine;R₁ is absent or is an amino terminal capping group of the peptidecompound; Xaa₃ is absent or Gly; Xaa₄ is absent, Asp, or Phe; Xaa₅ isabsent, Ala, or Phe; Xaa₆ is absent or Ala; R₂ is absent or is a carboxyterminal capping group of the peptide compound; and wherein the peptidecompound upregulates expression of a gene encoding an antioxidativeenzymes; andR₁ Xaa₁ Xaa₂ Xaa₃ R₂,wherein Xaa₁ is Asp, Asn, Glu, Gln, Thr, or Tyr; Xaa₂ is absent or anyamino acid; Xaa₃ is Asp, Asn, Glu, Thr, Ser, Gly, or Leu; R₁ is absentor is an amino terminal capping group; R₂ is absent or is a carboxyterminal capping group; and wherein the peptide compound upregulatesexpression of a gene encoding an antioxidative enzyme.

Preferred dietary supplement compositions of the invention may compriseone or more peptide compounds that upregulate expression of at least onegene encoding an antioxidative enzyme selected from the group consistingof:

(SEQ ID NO: 1) Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln,(SEQ ID NO: 2) Gln Thr Leu Gln Phe Arg, (SEQ ID NO: 13)Glu Thr Leu Gln Phe Arg, (SEQ ID NO: 14)Gln Tyr Ser Ile Gly Gly Pro Gln, (SEQ ID NO: 15)Ser Asp Arg Ser Ala Arg Ser Tyr, (SEQ ID NO: 12)Ser Lys Met Thr Leu Thr Gln Pro, (SEQ ID NO: 13)Met Thr Leu Thr Gln Pro, (SEQ ID NO: 16)Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu, (SEQ ID NO: 6)Asp Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 4) Asp Gly Asp Gly Asp,(SEQ ID NO: 8) Asn Gly Asn Gly Asp Phe Ala, (SEQ ID NO: 17)Asn Gly Asn Gly Asp, (SEQ ID NO: 7) Asp Gly Asn Gly Asp Phe Ala,(SEQ ID NO: 18) Asp Gly Asn Gly Asp, (SEQ ID NO: 9)Asn Gly Asp Gly Asp Phe Ala, (SEQ ID NO: 19) Asn Gly Asp Gly Asp,(SEQ ID NO: 20) Asn Gly Asp Gly, (SEQ ID NO: 5) Asp Gly Asp Gly Phe Ala,(SEQ ID NO: 21) Asn Gly Asn Gly Phe Ala, (SEQ ID NO: 22)Asp Gly Asn Gly Phe Ala, (SEQ ID NO: 23) Asn Gly Asp Gly Phe Ala,Asp Gly Asp, Asn Gly Asn, Asp Gly Asn, Asn GlyAsp, Asn Ser Thr, Phe Asp Gln, Met Thr Leu, MetThr Asp, Met Thr Asn, Met Thr Thr, Met Thr Glu,Met Thr Gln, Thr Val Ser, Leu Thr Gln, Leu ThrGly, Leu Thr Tyr, Asp Gly, Asn Gly, Glu Gly, GlnGly, Glu Ala, Gln Ala, Gln Gly, Asp Ala, and Asn Ala.A particularly preferred peptide compound useful in manufacturingdietary supplement formulations of the invention comprises the aminoacid sequence Asp Gly.

Dietary supplement compositions of the invention may also comprise oneor more peptide compounds described herein that have an amino terminalcapping group and/or a carboxy terminal capping group. Preferably, theamino terminal capping group is selected from a group consisting of areduced or oxidized lipoic acid moiety (Lip), a glucose-3-O-glycolicacid (Gga) moiety, 1 to 6 lysine residues, 1 to 6 arginine residues, anacyl group having the formula R₃—CO—, where CO represents a carbonylgroup and R₃ is a saturated or an unsaturated (mono- or polyunsaturated)hydrocarbon chain having from 1 to 25 carbons, and combinations thereof.Even more preferably, the amino terminal capping group is the acyl groupthat is an acetyl group, a palmitoyl group, or a docosahexaenoic acidgroup (DHA). In another preferred embodiment, a peptide compound presentin a dietary supplement of the invention comprises a carboxy terminalcapping group selected from the group consisting of a primary orsecondary amine.

Compositions containing one or more endogenous peptide compoundsdescribed herein may be purified from a natural source using variousmethods and protocols available in the art, such as fractionation bycentrifugation, concentration, gel filtration chromatography, organicsolvent extraction, etc. Such methods are employed by those skilled inthe art to obtain a composition purified from an original naturalsource, such as green velvet antler of deer, which is commerciallyavailable as a powder, or wuzi yanzong herbal mixture, which iscommercially available as a dry or liquid herbal mixture. The desiredpurified composition will typically be enriched (more concentrated) foran endogenous peptide compound described herein. Such a purifiedcomposition may be marketed as an oral dietary supplement.Alternatively, a natural source purified compositions may also becombined with an exogenously provided, synthetically produced peptidecompound described herein to produce a dietary supplement. Dietarysupplement compositions of the invention may further contain othermarketable ingredients of interest, such as lazaroids, vitamins,enzymes, and peptides purported to provide a benefit to health orwell-being of the individual who ingests them. In addition, dietarysupplement compositions of the invention may also comprise one or morebinders, fillers, powders, silica, or other inert ingredients commonlyused in the dietary supplements or pharmaceutical industries to makemarketable forms of the supplement compositions, such as pills,capsules, lozenges, liquids, and syrups (see above section onpharmaceutical compositions). Unlike pharmaceuticals, however, thepeptide compounds and other ingredients used to make a dietarysupplement composition are typically not regulated or otherwisecontrolled by a federal regulatory agency.

Natural source purified compositions can be assayed for the presence ofone or more peptide compounds, and the activity to upregulate expressionof a gene encoding SOD and/or CAT assayed in vitro or in vivo inmammalian cells by any of the various methods described herein or theirequivalents. Such analysis provides the information that enables theconsistent manufacture of standardized lots of an oral dietarysupplement product, which contains an appropriate amount of a peptidecompound to provide the same or substantially the same lot to lotantioxidative activity to an individual who takes the supplement. Theability to consistently manufacture and deliver for sale lots of thesame oral supplement product having a standardized amount of aningredient of interest is highly desired in the dietary supplementsmarket where product consistency can play a critical role inestablishing consumer confidence and patronage for a particular product.

Additional aspects of the invention will be further understood andillustrated in the following examples. The specific parameters includedin the following examples are intended to illustrate the practice of theinvention and its various features, and they are not presented to in anyway limit the scope of the invention.

EXAMPLES Example 1 Synthesis of Representative Peptide Compounds

The following representative peptide compounds were synthesized by solidphase Merrifield synthesis (Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)):

CMX-9236D (SEQ ID NO: 26)([DHA]-Lys Lys Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu),CMX-9236 (SEQ ID NO: 26)(Lys Lys Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu), CMX-11540(SEQ ID NO: 16) ([Lip]-Asp Gly Asp Gly Asp Phe Ala Ile Asp Ala Pro Glu),CMX-99661 (SEQ ID NO: 5) ([Gga]Asp Gly Asp Gly Phe Ala), CMX-99655(SEQ ID NO: 5) ([Ac]Asp Gly Asp Gly Phe Ala), CMX-9960 (SEQ ID NO: 5)([Palm]Asp Gly Asp Gly Phe Ala), CMLX-9963 (SEQ ID NO: 6)([Ac]-Asp Gly Asp Gly Asp Phe Ala), CMX-9967 ([Ac]-Asp Gly Asp),CMX-9901 (SEQ ID NO: 27)(Lys Lys Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln), CMX-8933(SEQ ID NO: 24) (Lys Lys Glu Thr Leu Gln Phe Arg), CMX-9902(SEQ ID NO: 28) (Lys Lys Asp Gly Asp Gly Asp Phe Ala), CMX-99658(SEQ ID NO: 2) ([Ac]-Gln Thr Leu Gln Phe Arg), (SEQ ID NO: 2)Lys Lys Gln Thr Leu Gln Phe Arg, CMX-8933 (SEQ ID NO: 24)(Lys Lys Glu Thr Leu Gln Phe Arg),(described in U. S. Patent No. 5,545,719) CMX-1156 ([Lip]-Thr Val Ser),CMX-99647 ([Ac]Thr Val Ser), CMX-1152 (Asp Gly), CMX-1159([Lip]-Asp Gly), and CMX-99672(trifluoroacetic acid salt of dipeptide Asp Gly).Amino terminal capping groups are indicated by the bracketed groups“[DHA]-”, “[Lip]-”, and “[Ac]-”, which represent an allcis-docasahexaenoic acid moiety, a lipoic acid moiety, and an acetylmoiety, respectively, attached by acylation to the α-amino group of theamino terminal amino acid residue of the indicated peptide compounds(Shashoua and Hesse, Life Sci. 58:1347-1357 (1996)).

The peptides were synthesized using standard procedures. Briefly, thepeptides were synthesized using the solid phase Merrifield process(Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154 (1963)). This methodallows the synthesis of a peptide of a specific amino acid sequencebound on a polymeric resin. Each newly synthesized peptide was thenreleased from the resin by treating with trifluoroacetic acid (TFA). Theresultant trifluoroacetic acid peptide salt was purified by etherprecipitation according to standard procedures (see, E. Groos andMeienhofer, In The peptides, analysis, synthesis, biology, vol. 2(Academic Press, New York 1983)).

For N-terminal substituted peptides (i.e., peptides containing an acylamino terminal capping group), each peptide was synthesized with blockedside chains using solid phase Merrifield synthesis (see above). Thebound peptide was then treated with an equimolar amount of an anhydrideof one of the following acids: acetic acid, DHA, or lipoic acid, in thepresence of 4-dimethylamino pyridine under argon atmosphere. Thereaction was carried out for about three hours to obtain N-terminalcoupling. Evidence of complete N-terminal coupling was obtained prior topeptide isolation. This was established by monitoring the ninhydrinstaining properties of the resin bound peptides using standardprocedures (E. Kaiser, et al., Anal. Biochem., 34: 595-598 (1970)). TheN-terminal coupled (capped) peptide molecule was then released from theresin by treatment with TFA and purified by precipitation with coldether followed by HPLC using methanolic HCl (50:50) as the eluant. Thefinal peptide products were white solids after lyophilization.Structures were confirmed by amino acid analyses, by migration as asingle peak on HPLC, and molecular weight determinations by massspectrometry. For most uses, it was essential to completely remove TFAfrom the peptide compound. This was achieved by repeated dissolution ofthe peptide in glacial acetic acid followed by concentration in vacuo inrotary evaporator. Complete absence of TFA was established by massspectrometry.

Example 2 Upregulation of Superoxide Dismutase (SOD) and Catalase (CAT)in Mammalian Cells by Peptide Compounds CMX-9236, CMX-9963, and CMX-9967

Upregulation of SOD

The RT-PCR method (see, for example, Innis et al., PCR Protocols: AGuide to Methods and Applications, (Academic Press, San Diego, 1990))was used to investigate the upregulation of the specific mRNA that codesfor the enzyme superoxide dismutase (SOD).

Primary cortical cultures were obtained by growing newborn rat braincortical cells in Delbecco's modified Eagle medium supplemented with 25μg/ml of gentamycin and 10% fetal calf serum. The cells were isolatedfrom the E-21 cortex of rat brain, plated at a density of 1×10⁵ per mland grown to confluence within four to five days in an atmospherecontaining air and 5% CO₂ at 37° C. as described in Cornell-Bell et al.,Science, 247: 470-473 (1990) and Cell Calcium, 12: 185-204 (1991).Cultures were grown in 20 ml flasks as a monolayer and then exposed tovarious concentrations of peptides for studies of the effects ofpeptides on upregulation of genes for SOD and CAT and on thetransmigration of transcription factor AP-1 to cell nuclei.

Primary cultures of rat brain cortical cells were incubated with 100ng/ml of peptide compound CMX-9236 for durations of 0 to 48 hours. mRNAwas isolated from the cytoplasmic fraction of lysed cells according tostandard methods (Angel et al., Cell 49:729-739, 1987). The RNA wasincubated according to the RT-PCR protocol with two strands, 20nucleotides long, one for the sense and one from the antisense strand.The sequences were selected to be unique for superoxide dismutase-1(SOD-1), and to span one intron segment. These were demonstrated to beunique by the BLAST program system (Nucl. Acids Res. 25:3389-3402,1997). The sequence of the two probe segments of the oligo dT segmentsfor SOD are as follows: anti-sense strand, ATCCCAATCACTCCACAGGCCAAGC(SEQ ID NO:29), and sense strand, GAGACCTGGGCAATGTGACTGCTGG (SEQ IDNO:30). These span a sequence of 208 base pairs (bp) on the primary SODsequence. The mixture was then treated with reverse transcriptase toobtain the cDNA according to standard PCR methods. The cDNA was thenanalyzed by electrophoresis and separated according to sequence lengthson a non-denaturing 5% polyacrylamide gel. The electrophoreticallyseparated molecules were treated with ethidium bromide to staindouble-stranded DNA fragments. These were visualized by ultravioletillumination and photographed. The gels were then analyzed by a laserscanning fluorescence detector to quantitate the amount of messenger RNAfor the extent of upregulation of SOD message and its time course ofsynthesis as a function of stimulation by CMX-9236. Similar methods ofanalysis were used for other peptides.

FIG. 1A shows the results of the electrophoresis. Each lane contained aninternal marker for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 451bp), a housekeeper marker to ensure that the same amount of RT-PCR cDNAproduct was actually loaded in each lane. The results show that after 3hours of incubation with 100 ng/ml of CMX-9236, there was a 9-foldupregulation of SOD-1 mRNA transcripts. The gel also contained apositive control (Pos) in which cortical cell cultures were stimulatedwith 10 μg/ml of the peptide compound for 3 hours, showing a maximumdevelopment of SOD stimulation.

FIG. 1B shows a bar graph depicting quantitative analysis data of theupregulation of SOD mRNA. Note that within 3 hours, there is a 9-foldincrease in upregulation of the mRNA with 100 ng/ml of peptide CMX-9236.This stimulation returns to control levels within 24 hours. The resultsdemonstrate that CMX-9236 can upregulate the mRNA that codes forsuperoxide dismutase-1 (SOD-1). Filled bars indicate levels of SOD-1;open bars indicate levels of GAPDH.

Similar RT-PCR experiments showed that cultures of rat primary myocytesupon stimulation with 1-100 ng/ml of CMX-9236 resulted in an enhancedproduction of mRNA coding for SOD-1. The amount of upregulation of SODwas 3-fold for the 1 ng/ml, and rose up to 6-fold at the 10-100 ng/ml(FIGS. 2A and 2B). FIG. 2A shows the dose-response data for the effectof CMX-9236 on the pattern of mRNA synthesis in primary myocyte culturesafter a 3-hour incubation. The presence of a band at the region of thegel corresponding to 208 base pairs indicates that SOD is upregulated.FIG. 2B shows a bar graph depicting quantitative analysis dataindicating that the 10 ng/ml and 100 ng/ml doses of CMX-9236 produced anupregulation of about 6-fold in the level of SOD-1 transcripts. Filledbars indicate fold-increase in levels of SOD-1 transcripts; open barsindicate fold-increase in levels of GAPDH transcripts.

The results shown in FIGS. 1A, 1B, 2A, and 2B demonstrated that thepeptide compound is capable of upregulating SOD-1 mRNA in at least twodifferent tissues, i.e., brain and muscle.

Translation of SOD mRNA to SOD-1 Protein

Additional evidence for increased synthesis of SOD within stimulatedcells was obtained by studies of the effect of representative peptidecompounds on the pattern of protein synthesis. Time course and doseresponse studies on rat brain cortical cell cultures showed that thelevel of immunoreactive (i.e., anti-SOD antibody reactive) proteins thatwere synthesized in the cytoplasm increased as a function of treatmentwith the peptide compound CMX-9967. Rabbit polyclonal antibodies(Rockland, Inc., Gilbertville, Pa.) in a Western blot assay showed adose-dependent antibody binding to the electrophoretically separatedcytoplasmic SOD proteins on polyacrylamide gels (FIG. 3A). Specifically,FIG. 3A shows a Western blot containing a band migrating at 34 kDa (themolecular weight of SOD-1), and two lower molecular weight bandscorresponding to smaller components recognized by the anti-SOD-1antibody in cells from cultures incubated for 5 hours in the presence of10 and 100 ng/ml of the CMX-9967 peptide.

FIG. 3B shows a bar graph of the quantitative analysis of the dataplotted as fold-increase in SOD-1 protein as a function of dose of theCMX-9967 peptide. At least a 20-fold increase in the intensity ofantibody binding occurred at the position of the cytoplasmic SOD-1 (mol.wt. 34,000 daltons) and its lower molecular weight analogue in cellstreated for 5 hours with 10 and 100 ng/ml of CMX-9967. This represents alarge increase as compared to the published data for transgenic micewith an extra SOD gene insert where at most a 50% increase in SOD wasdetected (Murakami et al., Stroke, 28: 1797-1804 (1997); Ceballos-Picot,CR Seances Soc. Biol. Fil., 187: 308-323 (1993)). The DNA sequence ofsuch mice contains a second SOD gene insert. Thus, these data indicatethat a peptide compound of the invention can upregulate mRNA for SOD-1,which is translated into a protein that has the same immunoreactiveproperties as SOD.

Upregulation of mRNA for Catalase (CAT)

Peptide compounds of the invention may also upregulate mRNA for catalase(CAT). Experiments using the RT-PCR methods to detect upregulation ofCAT mRNA in primary rat cortical brain cultures treated with variousrepresentative peptide compounds, i.e., CMX-9236, CMX-9963, andCMX-9967. The catalase probe duplex consisted of a sense primer havingthe sequence GCCCGAGTCCAGGCTCTTCTGGACC (SEQ ID NO:31) and antisenseprimer having the sequence TTGGCAGCTATGTGAGAGCCGGCCT (SEQ ID NO:32)flanking a 95 bp region of the CAT DNA.

Primary rat brain cortical cell cultures were incubated with 100 ng/mlof the peptide compound CMX-9236 for 0, 0.5, 1, 2, 3, 6, 12, 24, and 48hours. The results of the RT-PCR method are shown in FIGS. 4A and 4B.FIG. 4A shows a gel of RT-PCR products. The upregulation of CAT mRNA wasmaximal at 3 hours after the addition of the peptide compound, and itdecreased back to control levels at 48 hours. GAPDH was the internalstandard in these experiments. FIG. 4B shows a bar graph of thequantitative analysis of the data plotted as fold-increase as a functionof hours of treatment. A 13-fold increase in CAT mRNA levels wasobserved (over control) of CAT mRNA occurred when primary rat braincortical cultures were incubated with 100 ng/ml of CMX-9236 for 3 hours.

FIGS. 5A and 5B show results that demonstrate that other peptidecompounds (i.e., CMX-9963 and CMX-9967) can also upregulate both CAT andSOD in primary rat cortical cultures. FIG. 5A shows a gel of RT-PCRproducts for cultures incubated for 3 hours with 0, 1, 10, and 100 ng/mlof CMX-9963 or CMX-9967. FIG. 5B shows bar graphs of the quantitativeanalysis data plotted as fold-increase as a function of dose for eachpeptide. The bar graphs in FIG. 5B show that rat primary cortical cellsincubated with 10 ng/ml of CMX-9963 showed a maximum increase of around10-fold and 7-fold for SOD-1 (black bars) and CAT mRNA (open bars),respectively, whereas cells incubated with CMX-9967 showed a maximumincrease of 6-fold for both SOD and CAT at concentrations of 10 ng/ml.GAPDH was the internal standard. These findings establish that the CMXpeptide compounds can promote the synthesis for two of the primaryendogenous antioxidative enzymes that can defend cells from the powerfuleffects of ROS and free radicals.

Upregulation of SOD in Rat Fibroblasts by Peptide Compounds CMX-9963 andCMX-9967

Rat fibroblasts were isolated from lungs of rat embryos (E-21) and grownin culture as for culturing rat brain cortical cells as described above.After five days in culture, confluent monolayers of fibroblasts wereobtained and used in dose-response studies with CMX-9963 or CMX-9967 invehicle buffer (Hanks balanced salt solution (“HBSS”, Life Technologies,Baltimore, Md.) containing 5% xylitol) at 0, 1, 5, 10, and 100 ng/ml.After incubation in the presence of peptide compound for 6 hours,cytoplasmic fractions were isolated from each culture.

Cytoplasmic proteins were isolated according to published methods (Adamset al., J. Leukoc. Biol., 62: 865-873 (1997)). The cell cultures werewashed once in phosphate buffer saline (PBS) containing 20 mM EDTA andthen suspended in 250 μl of freshly prepared lysis buffer (20 mM Hepes,pH 7.9, 10 mM KCl, 300 mM NaCl, 1 mM MgCl₂, 0.1% Triton X-100 nonionicdetergent, 20% glycerol, 0.5 mM dithiothreitol (DTT), freshlysupplemented with inhibitors as described in Adams et al., J. Biol.Chem., 77: 221-233 (2000)). The suspensions were then incubated for atleast 10 minutes on ice to lyse cells and then centrifuged (14,000×g for5 minutes at 4° C.) to pellet cell debris. The supernatant cytoplasmicfractions were removed and stored as aliquots at −80° C. for analysis.The protein concentrations of the cytoplasmic fraction varied within 2-6μg/μl.

The cytoplasmic proteins were separated by SDS-PAGE using 5 μg/lane onthe gels. The gels were processed for Western immunoblots basically asdescribed by Adams et al. (General Cellular Biochemistry, 77: 221-233(2000)) to measure upregulation of SOD. The Western blots were alsoanalyzed by laser densitometry to quantify SOD protein upregulation. Thecontrol for this experiment was an identical culture flask which wastreated with vehicle (buffer, no peptide compound).

Western blots showed that both CMX-9963 and CMX-9967 upregulated SODgene expression in rat fibroblasts. In particular, incubation with 10ng/ml of either CMX-9963 or CMX-9967 resulted in at least a 20-foldincrease in (upregulation of) SOD gene expression in rat fibroblastsrelative to untreated cells.

Example 3 Upregulation of AP-1 Transcription Factor in Mammalian Cellsby Peptide Compounds

Immediately prior to use, a 1.0 mg aliquot of a peptide compound wasdissolved in 1.0 ml of 5% xylitol in HBSS (Hanks' Balanced SaltSolution, Hanks and Wallace, Proc. Soc. Exp. Biol. Med. 71:196 (1949)),the pH neutralized with 0.1 N NaOH, and the solution filter sterilized.Each peptide compound migrated as a single peak on HPLC column (C-18).The structure of each peptide compound was confirmed by amino acidanalysis, and its molecular weight was verified by mass spectroscopy.

Nuclear extracts for electrophoretic mobility shift assays (EMSAs) wereprepared as described previously (Adams et al., J. Leukocyte Biol., 62:865-873 (1997)) using 1.0−2.0×10⁷ cells per sample. All buffers werefreshly supplemented with dithiothreitol (DTT, 0.5 mM), proteaseinhibitors: PMSF (0.5 mM), chymostatin, peptstatin-A, aprotinin,antipain, leupeptin (each at 1 μg/ml), and phosphatase inhibitors: NaF(10 mM), ZnCl₂ (1 mM), sodium orthovanadate (1 mM), and sodiumpyrophosphate (5 mM). Aliquots of the final dialyzates were stored at−80° C. and discarded after use.

NB2a cells (1.0−2.0×10⁷ per sample) were washed in 1×PBS, 20 mM EDTA,then resuspended in 250 μl of lysis buffer (20 mM HEPES, pH 7.9, 10 mMKCl, 300 mM NaCl, 1 mM MgCl₂, 0.1% Triton X-100, 20% glycerol) freshlysupplemented with DTT and inhibitors as described above. Suspensionswere incubated for at least 10 minutes on ice to lyse cells, thencentrifuged (14,000×g, 5 minutes, 4° C.) to pellet cell debris.Supernatant aliquots were stored at −80° C. and discarded after a singleuse.

Electrophoretic Mobility Shift Assays (EMSAs)

AP-1 transcription factor activation was assayed using anelectrophoretic mobility shift assay (EMSA), as described by Adams etal., J. Leukoc. Biol., 62:865-873 (1997)). Cultures of primary ratneurons (Cornell-Bell et al., Cell Calcium, 12: 185-204 (1991)) werestimulated for 3 hours with various concentrations (0, 1, 10, 100 ng/ml)of peptide CMX-9236. Nuclear extracts prepared as described above wereseparated by gel electophoresis on non-denaturing gels and subjected tothe EMSA procedure. This EMSA used an AP-1 synthetic duplex probe(Angel, P., 1987, Cell 49:729-739) having the sequence5′-CGCTTGATGACTCAGCCGGAA (SEQ ID NO:33) and its antisense copy(complement strand), which were end-labeled with P³² usingpolynucleotide kinase and (γ-P³²)-ATP. For the EMSA reaction, thelabeled probe (0.5 pmol) was mixed with 3 μg of nuclear extract proteinin a solution containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA,1 mM dithiothreitol, 5% glycerol, 0.02% β-mercaptoethanol, and 1 μg ofpoly-dI/dC (Pharmacia). Reaction mixtures were incubated at 25° C. for20 minutes to allow complete complex formation by the duplex with itsappropriate AP-1 protein. The mixture was then electrophoresed undernon-denaturing conditions through 4% polyacrylamide gels in 0.5×TBEbuffer (45 mM Trisma base, 45 mM boric acid, 1 mM EDTA). The gels weredried on 3 mm paper. Bands were visualized by autoradiography at −80° C.with one intensifying screen and quantified by laser densitometry. Theupregulation of AP-1 and the activation and upregulation process of AP-1was compared to control cultures.

FIG. 6A shows the autoradiogram of a gel of the EMSA procedure for theprimary rat neurons. Maximum upregulation was obtained when suchcultures were incubated with 100 ng/ml of peptide compound CMX-9236.FIG. 6B shows a bar graph of the quantitative analysis of the dataplotted as fold-increase as a function of dose of CMX-9236. The dataindicate that there was a 60-fold increase in the binding of the DNAduplex probe at the position of the electrophoretic migration of thecomplexes formed with the DNA probe and the c-Jun/c-Fos heterodimer andthe c-Jun/c-Jun homodimer forms of AP-1 in the gel; indicative ofupregulation of AP-1 (see, FIGS. 6A and 6B).

Probe Competition EMSA

Probe competition EMSAs were carried out as in the EMSAs describedabove, except that a non-radiolabeled (“cold”) duplex AP-1 oligomer (seeabove) or “cold” mutant AP-1 duplex oligomer comprising the nucleotidesequence CGCTTGAGACTTGGCCGGAA (mutant bases underlined, SEQ ID NO:34)and its complementary strand were added to the EMSA reactions andincubated for 20 minutes at 25° C. prior to addition of the ³²P-labeledprobe. Following radiolabeled probe addition, the incubation wascontinued at 25° C. for an additional 20 minutes prior toelectrophoresis. The molar excess (0×, 5×, 25×, 50×) of cold proberelative to the 0.5 pmol of radiolabeled probe eliminated binding of thelabeled duplex added to each electrophoretic lane. The cold mutant probewith mutations at the two underlined positions (TG) could not eliminatebinding. This signifies that the probe had the correct sequence and ahigh degree of specificity for AP-1 (see, FIG. 6C).

Improved EMSAs may now be carried out basically as described above,except that an AP-1 synthetic duplex probe comprising the nucleotidesequence CGCTTGATGA GTCAGCCGGA A (SEQ ID NO:35) and its antisense copy(complement strand) are used for the standard assay and a mutant AP-1duplex oligomer comprising the nucleotide sequence CGCTTGATGAGTTGGCCGGAA (mutant bases underlined, SEQ ID NO:36) and itscomplementary strand are used for the probe competition assay.

A number of investigations have demonstrated that both nerve growthfactor (NGF) (Hsu et al., Stroke, 24 (suppl. I): I-78-I-79 (1993)) andbrain derived neurotrophic factor (BDNF) (Schabitz et al., J. Cereb.Blood Flow Metabol., 17: 500-506 (1997)), which are high molecularweight proteins than the peptide compounds described here, can stimulateneuronal growth by a mechanism that involves the activation(upregulation of expression) of the gene encoding transcription factorAP-1. The data indicate that the peptide compounds described herein alsoupregulate expression of the gene encoding AP-1 (see, FIGS. 6A, 6B, and6C).

Primary cortical cell cultures were grown to confluence and used in invitro cultures for determining the effects of various representativepeptide compounds on upregulation of AP-1. Stimulation of AP-1 factorwas found to increase in a dose-dependent manner, increasing by about15-fold up to a maximum of 60-fold after a 3-hour incubation at 35.5° C.with 1, 10, or 100 ng/ml concentrations of the peptide (FIG. 6A).Extracts were prepared from the nuclei isolated from the primary culturehomogenates, purified, and analyzed for the presence of specifictranscription factors using the electrophoretic mobility shift assay(EMSA) method. The binding of ³²P-labeled duplex DNA probes specific foractivator protein-1 (AP-1) or nuclear factor κB (NF-κB) was analyzed andquantified by autoradiography. After a 3-hour incubation of the cultureswith 1 ng/ml of the peptide CMX-9236 there was a 15-fold increase ofAP-1 relative to unstimulated control cultures. Incubations with 100ng/ml of the peptide typically increased the level of AP-1 by 60-fold.This suggests that the peptide compounds described herein are able toaffect the cascade of biochemical events that cause the phosphorylationof AP-1 and its translocation to the cell nuclei to increase c-Fos andthe upregulation of AP-1-dependent gene expression. Thus, a smallpeptide compound that is less than 20 amino acids long can simulate theproperties of large growth factors such as NGF and BDNF, which exist asdimers of protein chains with molecular weights of 13,259 and 13,500,respectively. The data suggest that the peptide compounds describedherein are capable of activating genes that may be involved in braincell growth. Such a mechanism has been previously demonstrated to blockapoptosis, reversing programmed cell death in the nematode (Horvitz etal., Cold Spring Harbor Symposium Quant. Biol., 111:377 (1994)) AndMammalian Nervous System (Yuan et al., Cell 75:641 (1993).

In control experiments (a negative control), the amino terminal cappinggroup and blood-brain barrier transmigration facilitator DHA, alone, didnot activate AP-1. However, peptide compounds without this DHA aminoterminal capping group could activate AP-1 at an equivalent molarconcentration to the DHA-coupled peptide compound. This indicates thatthe stimulation of AP-1 activity by the peptide compounds describedherein depends on the peptide sequence.

The specificity of the interaction of the DNA probe with AP-1 wasdemonstrated in two additional types of control experiments. A 5-foldmolar excess of non-radioactive AP-1 probe was found to completely blockthe formation of AP-1-³²P-DNA complex (see, FIG. 6C), while AP-1 withtwo errors in its sequence completely lost its capacity to formcomplexes even when used at a 50-fold molar excess relative to theradiolabeled probe (see, FIG. 6C). These results demonstrated a highdegree of specificity of the AP-1 probe interaction and validated theEMSA assay.

The data indicate that the peptide compounds can activate AP-1 geneexpression in neuronal cells. They demonstrate that a small peptidecompound can have properties similar to those of a large neurotrophicprotein factor, such as BDNF or NGF, which stimulate neuronal growth viaactivation of AP-1. Further evidence in support of such a concept is thefinding that an inhibition of activation of AP-1 correlates with eventsthat lead to neuronal cell death (Tabuchi et al., J. Biol. Chem., 271:31061-31067 (1996)). Upregulation of AP-1 seems to correlate with theprocess of cell growth, and its down-regulation seems to correlate withthe process of cell death. In one other control experiment, peptidecompounds did not promote the upregulation of the transcription factorNF-κB. This is a transcription factor that is associated with immuneresponses, i.e., not related to neuronal growth (Adams et al., J.Leukoc. Biol., 62: 865-873 (1997)). Such a result has also been reportedin literature for NGF, which is found to activate AP-1, but not NF-κB inPC12 cells (Tong and Perez-Polo, J. Neurosci. Res., 45: 1-12 (1996)).

Example 4 In Vivo Pharmacological Activity of Peptide CompoundsCMX-9236, CMX-9236D, CMX-9967, and CMX-9902

The in vivo neuroprotective effects of CMX-9236 were investigated inboth temporary and permanent occlusion stroke models in Sprague-Dawleyrats using an intraluminal suture of the middle cerebral artery (MCA)occlusion method (Zea Longa et al., Stroke, 20: 84 (1989)). Briefly, a4-0 silicone-coated suture was inserted through the right common carotidartery to block the MCA orifice. In the temporary model, CMX-9236 (6.2mg/kg/hr) or vehicle was administered via the femoral vein at 30 minutesafter the start of a 2-hour occlusion for a 4-hour continuous infusion;the rats were then reperfused by withdrawing the suture at 90 minutesafter the MCA occlusion. All experiments were performed in a blinded andrandomized manner, and rectal temperature was maintained at 37° C. Theanimals were sacrificed, and their brains were removed, sectioned intosix 2-mm-thick coronal slices and stained with2,3,5-triphenyltetrazolium chloride solution (Bederson et al., Stroke,17:1304 (1986)) to visualize the extent of brain damage for thecalculation of the corrected hemispheric infarct volumes (Nagasawa andKogure, Stroke, 20:1037 (1989); Li et al., J. Cereb. Blood Flow Metab.,17:1132 (1997)). In rats (n=10) treated with CMX-9236, the mean±S.E. forthe corrected infarct volume was found to be 117.3±17 mm³, as comparedto 178.8±11 mm³ for the vehicle-treated controls (n=10). This representsa significant reduction of infarct size (35±5%, p=0.01, student t-test)for the CMX-9236-treated group in the temporary occlusion model. Therewas also a 58+11% improvement in the neurological scoring (Minematsu etal., Neurology, 42:235 (1992)) for the peptide-treated group versuscontrols at the end of 24 hours.

In the permanent occlusion stroke model, the blood flow to the MCAterritory was blocked for the total 24-hour period. A continuous i.v.infusion (0.5 ml/hr) of the CMX-9236 peptide (2.04 mg/kg/hr) or vehiclewas initiated at 30 minutes after occlusion for 6 hours, followed by abolus i.v. infusion of CMX-9236 (4.0 mg/kg in 0.5 ml delivered over 10minutes) or vehicle at 12 hours after occlusion. The corrected infarctvolumes were 127.5±18 mm³ versus 216±18 mm³ for drug-treated animals andcontrols, respectively. This represents a significant decrease (41±5%,p=0.003, student t-test) in infarct size for the drug-treated group ascompared to the vehicle-treated control group (n=10 per group) in thepermanent model, indicating a substantial rescue of brain tissue. Thesefindings indicate that CMX-9236 has neuroprotective propertiespost-trauma in vivo, reducing the brain damage generated by cerebralischemia. Table 2 shows the results for different CMX peptides using theMCA permanent occlusion test.

TABLE 2 Effects of CMX Peptides on Infarct Size and NeurologicalBehavior of Rats Using the Permanent MCA 24-Hour Occlusion MethodPercent Dose Percent Neurological Compound (mg/hr) n Rescue ^(a) RescueCMX-9236D 0.03 7 48 ± 12 50 ± 18 (DHA-capped peptide) CMX-9236 0.05 4 23± 7 35 ± 8 (uncapped peptide) CMX-9967 0.05 4 20 ± 6 40 ± 10 CMX-99020.05 8 29 ± 7 35 ± 12 ^(a) Percent rescue denotes the percent decreasein infarct size in comparison to identical treatment of controls (n = 8)with vehicle alone.

Example 5 Activities of Other Representative Peptide Compounds

Certain activities have been demonstrated for other representativepeptide compounds using one or more the assays described in theproceeding Examples. CMX-9901 and CMX-8933 peptide compounds upregulatedAP-1 and provided a positive neuroprotective effect in the in vivopermanent MCA assay. CMX-9902 upregulated SOD proteins and increasednuclear migration of AP-1 in culture in vitro studies.

Example 6 In Vivo and In Vitro Pharmacological Activity of RelatedDipeptide Compounds CMX-1152 (Asp Gly) and CMX-99672 (TFA Salt of AspGly)

In vivo experiments were carried out in Sprague-Dawley rats (300-325 g)with solutions of CMX-1152 or CMX-99672. The animals were injectedintravenously (iv) via the tail vein with a peptide compound. Eachanimal received three injections, one hour apart (i.e., 0.3 ml of thepeptide compound at a concentration of 10 μg/ml in normal saline for atotal dose equivalent to 9 mg peptide compound/kg body weight. Theanimals were sacrificed by decapitation at 6, 12, 24, 48, and 72 hourspost injection and dissected to isolate brain, liver, heart, kidney,lung organs, which were frozen at −70° C. for subsequent analysis. Inaddition, 2 ml samples of whole blood were taken from each animal. Half(1 ml) of each sample was centrifuged to remove nuclei and cellmembranes to yield plasma. The remaining half was stored frozen as wholeblood at −70° C.

Western Immunoblots Using Antiserum to Human SOD and CAT Enzymes

Each tissue was thawed and homogenized in a Down's homogenizer using tenvolumes of homogenizer buffer (see, Adams et al., General CellularBiochemistry, 77: 221-233 (2000); buffer as described in Adams et al.,J. Leukoc. Biol., 62: 865-875 (1967)) to obtain a crude cytoplasmicfraction. The tissue homogenates were centrifuged (14,000×g for 5minutes at 4° C.) to yield the supernatant purified cytoplasmic proteinfractions as described in Adams et al. (J. Cell. Biochem., 77: 221-233(2000)). A 10 μg sample of each protein fraction was then separated bySDS polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed for SODand CAT content by Western blot assay.

Control for measurement of unstimulated levels of SOD and CAT wereobtained from two vehicle only (i.e., no peptide compound) injected ratsthat were sacrificed at 24 and 72 hours post injection. Both hadessentially the same unstimulated levels of SOD and CAT. Standardquantities of each cytoplasmic fractions (10 μg) were loaded on a laneof a gel for electrophoretic separation and Western immunoblot analysis(Adams et al., J. Cell. Biochem., 77: 221-233 (2000)). The stained gelswere photographed and scanned by laser densitometry to quantifyintensities in comparison to enzyme levels for control vehicle treatedrats.

Table 3 shows the upregulation data for SOD in various rat organscompared to control animals that received only the injection vehiclewithout peptide compound. The data show that administration of thepeptide compound CMX-1152 resulted in approximately a four to five-foldupregulation in SOD production in brain, heart, lung, and blood relativeto the control, and an approximately two-fold upregulation in SODproduction in liver and kidney. These results indicate that the peptidecompound CMX-1152 is active in vivo and that it is active in every majortissue and organ. Thus, the peptide compound CMX-1152 can upregulate SODin whole animals. Similar results were obtained for CAT (data notshown). These findings demonstrate the potential of CMX-1152 for use asa treatment that promotes the defense of the whole organism against ROSand free radicals. Accordingly, the body-wide, substantial antioxidativeactivity generated by the peptide compound CMX-1152 qualifies thispeptide compound as a particularly, well suited anti-aging candidatecompound that may be developed for use as a “healthy life expectancy”drug.

TABLE 3 In Vivo Upregulation of Superoxide Dismutase (SOD) inSprague-Dawley Rats After a 6-Hour Treatment with CMX-1152 at a Dose of10 mg/kg Organ SOD Activity (% of Control*) Brain 520 Heart 550 Liver200 Lung 520 Kidney 200 Plasma 420 Whole Blood 490 *Control levels =100%; injection of normal saline (vehicle)

Additionally, studies of the time course of the in vivo upregulation ofSOD and CAT by administration of the peptide compound CMX-1152 persistedfor a substantially longer period in tissues than in tissue not exposedto CMX-1152. The in vivo data given in Table 3 compare SOD levels forCMX-1152 versus vehicle (saline) treated controls. The control valuesfor each tissue remained constant as a function of time.

Additional Studies of CMX-1152 and CMX-99672

CMX-99672 and CMX-1152 peptide compounds comprise the same Asp-Glydipeptide. CMX-99672 is the trifluoracetic acid salt form and CMX-1152is the acetate salt form of the dipeptide. In this study, CMX-99672 wasonly used in the describe in vitro tissue culture experiments, whereasCMX-1152, as a purified acetate salt form that is free oftrifluoroacetic acid, was used in all in vivo experiments.

Tissue culture experiments were carried out using primary cultures ofrat brain cortical cells as previously described above in Example 2.Primary cultures from embryonic rat brain isolated at E-21 (21 dayembryos) and incubated for five hours with various doses: 1, 10, 100ng/ml of CMX-99672. Cytoplasmic proteins were isolated and analyzed forupregulation of SOD and CAT by Western immunoblots as described above.Western blots showed upregulation of SOD and SOD-related protein byCMX-99672. The Western blot was scanned to quantify the fold-increase inSOD production in cell cultures treated with the CMX-99672 peptidecompound. Exposure to CMX-99672 resulted in an approximately 30-foldincrease in SOD and 20-fold increase in SOD-related protein. Comparabledata was obtained for CAT.

These data indicate that the dipeptide Asp-Gly is highly effective atsubstantially increasing the anti-oxidative activity in cells andtissues of a mammal, and especially in the cells and tissues of thecentral nervous system. Again, such data indicate that this simpleAsp-Gly dipeptide compound is a candidate compound for use incompositions and methods to counteract the effects of ROS and other freeradicals, whether generated by the aging process, disease, or drugtreatments.

Example 7 In Vitro Study of Peptide Compounds CMX-99658 and CMX-8933

Two other peptide compounds CMX-99658 ([Ac]-Gln Thr Leu Gln Phe Arg)(SEQ ID NO:2), and CMX-8933 (Lys Lys Glu Thr Leu Gln Phe Arg) (SEQ IDNO:24) (described in U.S. Pat. No. 5,545,719). The essential differencebetween these two compounds is not presence of the particular protectiveamino terminal capping group (i.e., acetyl or Lys Lys), but the presenceof the first amino terminal glutamine or glutamic acid in the corepeptide sequence. The two peptide compounds were tested for the abilityto upregulate SOD and CAT in rat primary cortical cells. Rat primarycortical cells were isolated from E-21 rat embryos as described above inExample 4, except the cells were incubated with CMX-99658 or CMX-8933 at0.7, 7, and 70 ng/ml. Cells were incubated for 6 hours with a peptide orwith a control medium containing no peptide. SOD and CAT levels wereanalyzed by Western immunoblot using commercially available antibodiesto detect SOD and CAT (Rockland, Inc., Gilbertsville, Pa.).

The data indicated that CMX-8933 and peptide compounds comprising theGlu Thr Leu Gln Phe Arg (SEQ ID NO:13) amino acid sequence are preferredin various methods of the invention that rely upon the upregulation ofSOD and/or CAT gene expression to provide the levels of antioxidativeenzyme activities to counteract the generation of ROS and other freeradicals (e.g., due to aging, drug treatment, and disease).

Example 8 Upregulation of SOD and CAT in Rat Primary Cortical Culturesby Other Representative Peptide Compounds

Representative peptide compounds were tested at various doses andcompared for their ability to upregulate expression of genes for SODand/or CAT in rat primary cortical cultures basically as describedabove. Cultures were incubated with a peptide compound for 5 hours at37° C. Cytoplasmic protein fractions were prepared as described above.Cytoplasmic proteins were separated by gel electrophoresis and analyzedby Western immunoblots using antisera to SOD and CAT, respectively(Rockland, Inc., Gilbertsville, Pa.). The results are shown in Table 4(below).

TABLE 4 Upregulation of SOD and CAT by PeptideCompounds in Rat Primary Cortical Cultures Peptide Dose SOD CAT(CMX designation) (pmol/ml) (% Control) (% Control) None (Control) 0 100100 Asp Gly 6.7 2143 N.D. (CMX-1152) [Ac]Asp Gly Asp 28 1220 200(CMX-9967) [Ac]Thr Val Ser 6.7 2066 N.D. (CMX-99647) [Gga]Asp Gly Asp6.7 463 N.D. Gly Phe Ala (CMX-99661) (SEQ ID NO: 5) [Ac]Asp Gly Asp 8750 520 Gly Phe Ala (CMX-99655) (SEQ ID NO: 5) [Palm]Asp Gly Asp 6.7 527N.D. Gly Phe Ala (CMX-9960) (SEQ ID NO: 5) [Ac]Asp Gly Asp 6.7 1873 N.D.Gly Asp Phe Ala 28 N.D. 700 (CMX-9963) (SEQ ID NO: 6) Lys Lys Gln Thr 95350 400 Leu Gln Phe Arg (SEQ ID NO: 25) Lys Lys Asp Gly 6.7 466 N.D.Asp Gly Asp Phe 67 N.D. 652 Ala Ile Asp Ala Pro Glu (CMX-9236)(SEQ ID NO: 26) Control = normal saline; SOD = superoxide dismutase; CAT= catalase; [Ac]= acetyl; [Gga]3-O-glucose-glycolic acid; [Palm]=palniltoyl; N.D. = not determinedThe above data demonstrate that the peptides are capable of upregulatingantioxidative enzymes, i.e., SOD and/or CAT. Peptide compoundscomprising the amino acid sequence Asp Gly Asp Gly Phe Ala (SEQ ID NO:5)were able to upregulate both SOD and CAT to essentially equal levels.Such equipotent activity for upregulating both SOD and CAT indicatesthat this peptide is particularly preferred for providing adequaterelative levels of the complementary antioxidative enzyme activities ofSOD and CAT to counteract oxidative stress produced from a variety ofsources and conditions, as well as to effectively detoxify hydrogenperoxide generated by SOD activity on super oxide anions.

Example 9 Preparation of an Active Fraction from Green Velvet Antler(GVA) and Formulation of Nutraceutical Compositions from an AnimalSource

Natural Source Purification, Analysis, and Formulation of NutraceuticalCompositions

Five grams of the raw GVA dry powder from Qeva, Inc. (Calgary, Ontario,Canada), were extracted with 100 ml of water at room temperature for 30minutes. The water soluble components (“GVAW”) were then separated fromthe insoluble residue by centrifugation for 30 minutes at 5,000×g. Theresidue was further extracted by re-suspension in 50 ml of water andstirring for additional 30 minutes. The mixture was then re-centrifuged(30 minutes at 5,000×g) and the supernatants from the two extracts werecombined to give a crude yellow extract. This was re-centrifuged at10,000×g for 30 minutes at room temperature. The supernatant fractionwas removed and sterilized by filtration through a Millipore filter (0.2μm pore size) to give a clear yellow solution. This clear yellowsolution was then concentrated to 10-20 ml in a rotary evaporator at 30°C. under mild vacuum, and lyophilized to give the fraction GVAW as abrown, fluffy powder (yield 15-20%). This fraction contains an activepeptide that can up-regulate SOD (see Table 5) in primary rat braincortical cultures as described above.

Additional purification of the GVAW fraction was carried out by columnchromatography using Biogel (PD-10) from Bio-Rad Laboratories (Hercules,Calif. 94547). This separated the peptides with a molecular weight (MW)higher than 6,000 daltons from those with a MW of less than 6,000daltons to give two fractions: GVA +6 and GVA −6, respectively, and togive yields (based on raw material) of lyophilized products of 8-10% and3-6%, respectively.

GVA −6 contained a concentrate of an active GVA peptide. Analysis bythin layer chromatography on silica gel flexible sheets (J. T. BakerInc., Phillipsturg, N.J.) using ethanol/ammonium hydroxide (70/30) asthe eluant showed the presence of many peptide components that includedone that had identical migration properties to CMX-152 (Asp Gly).Additional confirmation was obtained by mass spectroscopy that showedthe presence of two components that have MWs of 236 and 190corresponding to the disodium salt and the free acid form of Asp Gly,respectively. Amino acid analysis of the GVA −6 fraction showed thepresence of the amino acids Asp and Gly in equimolar amounts in themixture. GVA −6 also had the property of up-regulating SOD in rat brainprimary cortical cultures. It was at least 3,000 times more active thanthe GVA +6 fraction (see Table 5, below).

TABLE 5 SOD Dose Upregulation Fraction (ng/ml) (% Control) Control — 100GVAW 100 250 GVA −6 10 220 GVA −6 100 330 GVA +6 100 105 GVA +6 1000 132DG (CMX-1152) 1 170 DG 10 330 DG 100 420

Both GVAW and GVA −6 fraction were augmented with pure synthetic Asp Glypeptide to obtain a therapeutic level of SOD up-regulation properties ina standard assay using rat brain primary cultures. Typical formulationswill have the necessary amount of CMX-1152 to obtain an approximately1.3 to 10.0-fold up-regulation of SOD in plasma and blood samples.

Alternative Method of Preparing GVA −6 Fraction

The column chromatography step in the above method was replaced bydirect extraction of GVAW fraction with 100% methanol. Here the highmolecular weight components formed a precipitate, which was thenseparated to yield clear filtrate which, after treatment with activecharcoal, produced a colorless solution. The colorless solution yieldeda white solid after lyophilization. This had a similar composition tothat of GVA −6 described above.

Example 10 Preparation of an Active Fraction from a Plant Source andFormulation of Nutraceutical Compositions

Similar methods as described in Example 9 were used to obtain an activefraction from Wu, Zi, Yan, Zong, Wan herbal mixtures (see, e.g., Wang etal., Chung Kuo Chung H is I Chieh Ho Tsa Chih 12: 23-25 (1992), study ofwuzi yanzong liquid; Huang et al., Chung Kuo Chung Yao Tsa Chih 16:414-416, 447 (1991), study of fufang wuzi yanzong pills) to obtainformulations that have a standard level of SOD up-regulation properties.Typical formulations for nutraceutical compositions will be converted toa standard potency level by adding pure synthetic CMX-1152 to obtain SODup-regulation of approximately 1.3 to 10-fold up-regulation of SOD inplasma and blood samples.

Various amino acid and nucleotide sequences referred to herein and theircorresponding sequence identification numbers (SEQ ID NO:) are listed inTable 6, below. Variable amino acids (Xaa) present in some thesesequences are described in more detail above.

TABLE 6 Sequences and Corresponding Sequence Identification NumbersSequence  Identification Sequence NumberGln Tyr Lys Leu Gly Ser Lys Thr Gly SEQ ID NO: 1 Pro Gly GlnGln Thr Leu Gln Phe Arg SEQ ID NO: 2 Xaa₁ Gly Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆SEQ ID NO: 3 Asp Gly Asp Gly Asp SEQ ID NO: 4 Asp Gly Asp Gly Phe AlaSEQ ID NO: 5 Asp Gly Asp Gly Asp Phe Ala SEQ ID NO: 6Asp Gly Asn Gly Asp Phe Ala SEQ ID NO: 7 Asn Gly Asn Gly Asp Phe AlaSEQ ID NO: 8 Asn Gly Asp Gly Asp Phe Ala SEQ ID NO: 9Xaa₁ Xaa₂ Met Thr Leu Thr Gln Pro SEQ ID NO: 10 Met Thr Leu Thr Gln ProSEQ ID NO: 11 Ser Lys Met Thr Leu Thr Gln Pro SEQ ID NO: 12Glu Thr Leu Gln Phe Arg SEQ ID NO: 13 Gln Tyr Ser Ile Gly Gly Pro GlnSEQ ID NO: 14 Ser Asp Arg Ser Ala Arg Ser Tyr SEQ ID NO: 15Asp Gly Asp Gly Asp Phe Ala Ile Asp SEQ ID NO: 16 Ala Pro GluAsn Gly Asn Gly Asp SEQ ID NO: 17 Asp Gly Asn Gly Asp SEQ ID NO: 18Asn Gly Asp Gly Asp SEQ ID NO: 19 Asn Gly Asp Gly SEQ ID NO: 20Asn Gly Asn Gly Phe Ala SEQ ID NO: 21 Asp Gly Asn Gly Phe AlaSEQ ID NO: 22 Asn Gly Asp Gly Phe Ala SEQ ID NO: 23Lys Lys Glu Thr Leu Gln Phe Arg SEQ ID NO: 24Lys Lys Gln Thr Leu Gln Phe Arg SEQ ID NO: 25Lys Lys Asp Gly Asp Gly Asp Phe Ala SEQ ID NO: 26 Ile Asp Ala Pro GluLys Lys Gln Tyr Lys Leu Gly Ser Lys SEQ ID NO: 27 Thr Gly Pro Gly GlnLys Lys Asp Gly Asp Gly Asp Phe Ala SEQ ID NO: 28ATCCCAATCACTCCACAGGCCAAGC SEQ ID NO: 29 GAGACCTGGGCAATGTGACTGCTGGSEQ ID NO: 30 GCCCGAGTCCAGGCTCTTCTGGACC SEQ ID NO: 31TTGGCAGCTATGTGAGAGCCGGCCT SEQ ID NO: 32 CGCTTGATGACTCAGCCGGAASEQ ID NO: 33 CGCTTGATGACTTGGCCGGAA SEQ ID NO: 34 CGCTTGATGAGTCAGCCGGAASEQ ID NO: 35 CGCTTGATGAGTTGGCCGGAA SEQ ID NO: 36

Other variations and embodiments of the invention described herein willnow be apparent to those of ordinary skill in the art without departingfrom the scope of the invention or the spirit of the claims below.

All patents, applications, and publications cited in the above text areincorporated herein by reference.

1. An isolated peptide compound, wherein the amino acid sequence of thepeptide compound is Asp Gly Asp, Thr Val Ser, Glu Ala, or Asp Gly,wherein the peptide compound comprises an amino terminal capping groupand/or a carboxy terminal capping group.
 2. A composition comprising apeptide compound, wherein the amino acid sequence of the peptidecompound is Asp Gly Asp, Thr Val Ser, Glu Ala, or Asp Gly, wherein thepeptide compound comprises an amino terminal capping group and/or acarboxy terminal capping group; and the composition comprises apharmaceutically acceptable carrier.
 3. An isolated peptide compound,wherein the amino acid sequence of the peptide compound is Glu Gly, AspGly Asp Gly Asp (SEQ ID NO:4), Asp Ala, or Asp Gly Asp Gly Asp Phe Ala(SEQ ID NO:6), wherein the peptide compound comprises an amino terminalcapping group and/or a carboxy terminal capping group.
 4. A peptidecompound comprising a peptide, wherein the amino acid sequence of thepeptide compound is:R1 Xaa1 Xaa2 Xaa3 R2, wherein Xaa1 is Asp, Asn, Glu, Gln, Thr, or Tyr;Xaa2 is absent or any amino acid; Xaa3 is Asp, Asn, Glu, Thr, Ser, Gly,or Leu; R1 is absent or is an amino terminal capping group; R2 is absentor is a carboxy terminal capping group, wherein R1 and R2 are not bothabsent.
 5. A peptide compound comprising a peptide, wherein the aminoacid sequence of the peptide compound is:R1 Xaa1 Xaa2 Xaa3 R2, wherein Xaa1 is Asp, Asn, Gln, Thr, or Tyr; Xaa2is absent or any amino acid; Xaa3 is Asp, Asn, Glu, Thr, Ser, Gly, orLeu; R1 is absent or is an amino terminal capping group; R2 is absent oris a carboxy terminal capping group, wherein R1 and R2 are not bothabsent.
 6. The peptide compound of any of claims 1, 3, 4, and 5, whereinthe amino terminal capping group is selected from the group consistingof 1 to 6 lysine residues; 1 to 6 arginine residues; a mixture ofarginine and lysine residues ranging from 2 to 6 residues, a urethane, aurea, a glucose-3-O-glycolic acid group; an acyl group containing asaturated or unsaturated, branched or unbranched, hydrocarbon chain; orcombinations thereof.
 7. The peptide compound of claim 6, wherein theacyl group is an acetyl group; a palmitoyl group; a lipoyl group; adocosahexaenoyl group; a capryloyl group; a caproyl group; a lauroylgroup; a myristoyl group; a palmitoleoyl group; a stearoyl group; aoleoyl group; a vaccenoyl group; a linoleoyl group; an alpha-linolenoylgroup; eleostearoyl group; a beta-linolenoyl group; a gondoyl group; adihomo-gamma-linolenoyl group; a arachidonoyl group; a eicosapentaenoylgroup; a docosenoyl group; a docosatetraenoyl group; a docosapentaenoylgroup; a docosapentaenoyl group; or a nervonoyl group.
 8. The peptidecompound of any of claims 1, 3, 4, and 5, wherein the carboxy terminalcapping group is selected from the group consisting of an amino grouplinked to the carboxy terminal carbonyl of the peptide compound aminoacid sequence by an amide bond; an aliphatic alcohol linked to thecarboxy terminal carbonyl of the peptide compound amino acid sequence byan ester bond; or an aromatic phenolic derivative linked to the carboxyterminal carbonyl of the peptide compound amino acid sequence by anester bond.
 9. The isolated peptide compound of claim 1, wherein theamino acid sequence of the peptide compound is Asp Gly Asp.
 10. Theisolated peptide compound of claim 1, wherein the amino acid sequence ofthe peptide compound is Thr Val Ser.
 11. The isolated peptide compoundof claim 1, wherein the amino acid sequence of the peptide compound isGlu Ala.
 12. The isolated peptide compound of claim 1, wherein the aminoacid sequence of the peptide compound is Asp Gly.
 13. The composition ofclaim 2, wherein the amino terminal capping group is selected from thegroup consisting of 1 to 6 lysine residues; 1 to 6 arginine residues; amixture of arginine and lysine residues ranging from 2 to 6 residues, aurethane, a urea, a glucose-3-O-glycolic acid group; an acyl groupcontaining a saturated or unsaturated, branched or unbranched,hydrocarbon chain; or combinations thereof.
 14. The peptide compound ofclaim 13, wherein the acyl group is an acetyl group; a palmitoyl group;a lipoyl group; a docosahexaenoyl group; a capryloyl group; a caproylgroup; a lauroyl group; a myristoyl group; a palmitoleoyl group; astearoyl group; a oleoyl group; a vaccenoyl group; a linoleoyl group; analpha-linolenoyl group; eleostearoyl group; a beta-linolenoyl group; agondoyl group; a dihomo-gamma-linolenoyl group; a arachidonoyl group; aeicosapentaenoyl group; a docosenoyl group; a docosatetraenoyl group; adocosapentaenoyl group; a docosapentaenoyl group; or a nervonoyl group.15. The composition of claim 2, wherein the carboxy terminal cappinggroup is selected from the group consisting of an amino group linked tothe carboxy terminal carbonyl of the peptide compound amino acidsequence by an amide bond; an aliphatic alcohol linked to the carboxyterminal carbonyl of the peptide compound amino acid sequence by anester bond; or an aromatic phenolic derivative linked to the carboxyterminal carbonyl of the peptide compound amino acid sequence by anester bond.
 16. The composition of claim 2, wherein the amino acidsequence of the peptide compound is Asp Gly Asp.
 17. The composition ofclaim 2, wherein the amino acid sequence of the peptide compound is ThrVal Ser.
 18. The composition of claim 2, wherein the amino acid sequenceof the peptide compound is Glu Ala.
 19. The composition of claim 2,wherein the amino acid sequence of the peptide compound is Asp Gly. 20.The isolated peptide compound of claim 3, wherein the amino acidsequence of the peptide compound is Glu Gly.
 21. The isolated peptidecompound of claim 3, wherein the amino acid sequence of the peptidecompound is Asp Gly Asp Gly Asp SEQ ID NO:4).
 22. The isolated peptidecompound of claim 3, wherein the amino acid sequence of the peptidecompound is Asp Ala.
 23. The isolated peptide compound of claim 3,wherein the amino acid sequence of the peptide compound is Asp Gly AspGly Asp Phe Ala (SEQ ID NO:6).
 24. The peptide compound of claim 4,wherein R1 is a lipoyl group.
 25. The peptide compound of claim 5,wherein R1 is a lipoyl group.
 26. The peptide compound of claim 1,wherein the amino terminal capping group is present and the carboxyterminal capping group is absent.
 27. The peptide compound of claim 1,wherein the amino terminal capping group is [Lip].
 28. The isolatedpeptide compound of claim 1, wherein the peptide compound is [Lip]-GluAla.
 29. The composition of claim 2, wherein the peptide compound is[Lip]-Glu Ala.